Novel compounds for modulating cell proliferation

ABSTRACT

Novel styrylacrylonitrile compounds which are useful in treating a variety of cell proliferative disorders such as cancer are disclosed. The compounds are of the Formula I:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of provisionalapplication U.S. Serial No. 60/196,936, filed Apr. 13, 2000, thecontents of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

[0002] This invention relates to novel compounds which are useful fortreating a variety of cell proliferative disorders such as cancer.

BACKGROUND OF THE INVENTION

[0003] A wide range of growth factors coordinate cell proliferation anddifferentiation. Malignant cells arise as a result of a stepwiseprogression of events that include the unregulated expression of growthfactors or components of their signaling pathways. Tyrosinephosphorylation events initiated by receptor, cytoplasmic and nuclearkinases and regulated by phosphatases are central to these processes.Mutation, hyper-activation, translocation and overexpression of proteintyrosine kinases are all associated with tumorigenesis. In addition toincreasing proliferative rates and immortalizing cells, overexpressionof tyrosine kinases can lead to morphological transformation and causeanchorage independence, contributing to the promotion of migratoryability and possibly the induction of metastases.

[0004] Certain compounds with structures based upon mimicry of ATP orphosphotyrosine have been shown to be effective kinase inhibitors. Thosebased upon phosphotyrosine have been demonstrated to be the morespecific tyrosine kinase inhibitors. Because of their ability to inhibittyrosine phosphorylation, these compounds may alter cell responses togrowth factors or other process driven by tyrosine kinase activity,including unregulated growth as the result of tyrosine kinaseoverexpression, mutation, or translocation. Inhibition of tyrosinekinases occupying a central role in proliferative signaling pathways, orin pathways regulating cell cytoskeletal structure, even temporary orincomplete inhibition, may be sufficient to switch a cancerous cell froma proliferative cycle into programmed cell death, or apoptosis. Death byapoptosis is most often observed upon effective treatment with tyrosinekinase inhibitors.

[0005] Selective inhibition of specific tyrosine kinases offers a methodof targeting cancerous cell growth with a high degree of specificity andminimal toxicity to normally growing cells and tissues. Thus, specificinhibitors of tyrosine kinases have great potential as clinicalanti-cancer treatments. A number of small molecules which act astyrosine kinase inhibitors have been identified. For example, certainphenyl acrylonitrile compounds have been described as tyrosine kinaseinhibitors, effective to inhibit cell proliferation (see for example,U.S. Pat. Nos. 5,891,917, 5,217,999, 5,773,476, 5,935,993, 5,656,655,5,677,329 and 5,789,427).

[0006] Inhibition of tyrosine kinases offers one mechanism by which cellproliferation can be inhibited. One of skill in the art will appreciatethat other mechanisms of inhibition may also be involved

[0007] There is a need in the art to identify compounds that inhibitcell proliferation.

SUMMARY OF THE INVENTION

[0008] A number of novel compounds have now been identified that inhibitabnormal cell proliferation, for example cancer cell growth. Thecompounds do not inhibit the growth of normal cells.

[0009] Accordingly, the present invention includes compounds of FormulaI and salts, solvates and hydrates thereof:

[0010] wherein

[0011] R¹ and R² are each independently selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo;

[0012] R³ is selected from the group consisting of H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;

[0013] R⁴ is selected from the group consisting of C(X)R⁵, SO₃Ar, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), P(O)(OH)₂, P(O)(OC₁₋₆alkyl)₂, andC(NH₂)═C(CN)₂;

[0014] X is selected from O,S, NH and N-C₁₋₆alkyl;

[0015] R⁵ is selected from the group consisting of NH₂, OH,NH(CH₂)_(p)Ar, NH(CH₂)_(p)OH, (CH₂)_(p)OC₁₋₆alkyl, C₁₋₆alkyl,C₁₋₆alkoxy, NHNH₂, NHC(O)NH₂, NHC(O)C₁₋₆alkoxy, N-morpholino andN-pyrrolidino;

[0016] Ar is an aromatic or heteroaromatic group, unsubstituted orsubstituted with 1-4 substituents, independently selected from the groupconsisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo;

[0017] n is 0 to 4; and

[0018] p is 1-4.

[0019] The present invention further includes compounds of Formula IIand salts, solvates and hydrates thereof:

[0020] wherein

[0021] R¹ and R² are each independently selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo;

[0022] R³ is selected from the group consisting of H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;

[0023] Ar is an aromatic or heteroaromatic group, unsubstituted orsubstituted with 1-4 substituents, independently selected from the groupconsisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo;

[0024] R⁶ is selected from the group consisting of Ar, OH andOC₁₋₆alkyl;

[0025] X is selected from O and S;

[0026] n is 0-4; and

[0027] p is 1-4.

[0028] The present invention also provides compounds of Formula III andsalts, solvates and hydrates thereof:

[0029] wherein

[0030] R¹ and R² are each independently selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo;

[0031] R³ is selected from the group consisting of H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;

[0032] Ar is an aromatic or heteroaromatic group, unsubstituted orsubstituted with 1-4 substituents, independently selected from the groupconsisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo;

[0033] R⁷ is selected from the group consisting of OH, NH₂ andOC₁₋₆alkyl;

[0034] X is selected from O and S; and

[0035] n is 0-4.

[0036] According to another aspect of the present invention, there isprovided a pharmaceutical composition comprising a compound of theinvention and a pharmaceutically acceptable carrier or diluent.

[0037] In accordance with a further aspect of the present invention,there is provided a method for modulating cell proliferation, preferablyinhibiting cell proliferation comprising administering an effectiveamount of a compound of the invention to a cell or animal in needthereof. The invention also includes a use of a compound of theinvention to modulate cell proliferation, preferably inhibit cellproliferation. The invention further includes a use of a compound of theinvention to prepare a medicament to modulate cell proliferation,preferably inhibit cell proliferation.

[0038] In a preferred embodiment the present invention provides a methodof inhibiting the proliferation of a cancer cell comprisingadministering an effective amount of a compound of the invention to acell or animal in need thereof. The cancer cell treated may be any typeof cancer including a leukemia, a lymphoma, myeloma, metastaticcarcinoma, sarcoma or any other malignant transformation or any othermalignancy. The invention also includes a use of a compound of theinvention to modulate cancer cell proliferation, preferably inhibitcancer cell proliferation. The invention further includes a use of acompound of the invention to prepare a medicament to modulate cancercell proliferation, preferably inhibit cancer cell proliferation.

[0039] In another aspect, the invention provides a method of modulatingtyrosine kinase activity in a cell by administering an effective amountof a compound of the invention. In a further aspect, the inventionprovides a method of inhibiting tyrosine kinase activity in a cell byadministering an effective amount of a compound of the invention. Thepresent invention also provides a use of a compound of the invention tomodulate, preferably inhibit, tyrosine kinase activity. The presentinvention further provides a use of a compound of the invention toprepare a medicament to modulate tyrosine kinase activity, preferablyinhibit tyrosine kinase activity. It is appreciated that the inhibitionof cell growth by the compounds of the invention may be effected byother mechanisms.

[0040] Other features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The invention will now be described in relation to the drawingsin which:

[0042]FIG. 1 is a bar graph showing the effect of CR4 upon normal bonemarrow differentiation in culture.

[0043]FIG. 2 is a bar graph showing the killing of Philadelphia positiveacute lymphoblastic leukemia by low-dose CR4 in culture.

[0044]FIG. 3 is a bar graph showing the killing of Philadelphia positiveZ119 Acute lymphoblastic leukemia cells by low-dose CR4 in culture.

[0045]FIG. 4 is a bar graph showing the killing of AML-3 acute myeloidleukemia cells by low-dose CR4 in culture.

[0046]FIG. 6 is a bar graph showing the killing of Ly-MN lymphoma cellsby low-dose CR4 in culture.

[0047]FIG. 6 is a bar graph showing the killing of primary juvenilemyelo-monocytic leukemia cells by CR4 in culture.

[0048]FIG. 7 is a bar graph showing the killing of OCI-LY2 lymphomacells by low-dose CR4 in culture.

[0049]FIG. 8 is a bar graph showing the killing of Philadelphia positiveALL cells by CR17 and CR21 in culture.

[0050]FIG. 9 is a bar graph showing the killing of Philadelphia positiveALL cells by CR17 and CR21 in culture.

[0051]FIG. 10 is a bar graph showing the killing of Philadelphiapositive ALL cells by CR24 in culture.

[0052]FIG. 11 is a bar graph showing the killing of Philadelphiapositive ALL cells by CR19 in culture.

[0053]FIG. 12 is bar graph showing the effect of CR19 on normal bonemarrow differentiation in culture.

[0054]FIG. 13 is a bar graph showing the effect of CR24, CR17 and CR21on normal bone marrow differentiation.

[0055]FIG. 14 is a bar graph showing the effect of in vitro purging ofnormal bone marrow with CR4.

[0056]FIG. 15 is a bar graph showing the effect of in vitro purging ofZ119 acute lymphoblastic leukemia with CR4.

[0057]FIG. 16 is a bar graph showing the effect of In vitro purging ofOCI-Ly2 lymphoma cells with CR4.

[0058]FIG. 17 is a bar graph showing the effect of in vitro purging ofOCI-AML-3 acute meyloid leukemia cells with CR4.

[0059]FIG. 18 is a bar graph showing the effect of in vitro purging ofRamos B cell Burkitt's lymphoma cells with CR4.

[0060]FIG. 19 is a bar graph showing the killing of HuNS1 multiplemyeloma cells by low-dose CR4 in culture.

[0061]FIGS. 20A and B are graphs showing cell staining after in vivotreatment of Philadelphia positive acute lymphoblastic leukemia inNOD-SCID mice.

[0062]FIG. 21 is a bar graph showing that the effect of in vitro purgingof normal bone marrow with CR11.

[0063]FIG. 22 is a bar graph showing that the effect of in vitro purgingof Philadelphia positive acute lymphoblastic leukemia with CR11.

[0064]FIG. 23 is an autoradiograph which shows Philadelphia (Ph+) ALLlines Z119 and Z181 (5×10⁶ cells/point) immunoprecipitated with Bcr-Ablantibody.

[0065]FIG. 24 is an autoradiograph which shows Philadelphia (Ph+) ALLline Z119 (5×10⁶ cells/point) immunoprecipitated with Jak2 antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] Throughout this application, various publications, patents, andpublished patent applications are referred to by an identifyingcitation. The disclosures of the publications, patents, and publishedpatent applications referenced in this application are herebyincorporated by reference into the present disclosure in their entirety.

[0067] I. Definitions

[0068] The term “C₁₋₆alkyl” as used herein means, unless otherwisestated, straight and/or branched chain alkyl radicals containing fromone to six carbon atoms and includes methyl, ethyl, propyl, isopropyl,t-butyl and the like.

[0069] The term “C₁₋₆alkoxy” as used herein means, unless otherwisestated, straight and/or branched chain alkoxy radicals containing fromone to six carbon atoms and includes methoxy, ethoxy, propyoxyl,isopropyloxy, t-butoxy and the like.

[0070] The term “C₁₋₄alkyl” as used herein means, unless otherwisestated, straight andlor branched chain alkyl radicals containing fromone to four carbon atoms and includes methyl, ethyl, propyl, isopropyl,t-butyl and the like.

[0071] The term “C₁₋₄alkoxy” as used herein means, unless otherwisestated, straight and/or branched chain alkoxy radicals containing fromone to four carbon atoms and includes methoxy, ethoxy, propyoxyl,isopropyloxy, t-butoxy and the like.

[0072] The term “Ar” as used herein, means an unsubstituted orsubstituted aryl and/or heteroaryl group which, in the case ofheteroaryl, may contain up to two heteroatoms, wherein the constituentsare independently selected from the group consisting of OH, C₁₋₆alkyl,C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,NO₂, CF₃, OCF₃ and halo, and includes unsubstituted or substitutedphenyl, furyl, thienyl, indolyl, naphthyl, quinolyl and the like.

[0073] The term “halo” as used herein means halogen and includes chloro,flouro, bromo, iodo and the like.

[0074] The term “pharmaceutically acceptable salt” means an acidaddition salt or a basic addition salt which is suitable for orcompatible with the treatment of patients.

[0075] The term “compound of the invention” as used herein includes anycompound of the Formula I, II or III as defined herein (including allsalts, solvates or hydrates thereof) as well as a specific compounddesignated herein as CR1, CR2, CR3, CR4, CR5, CR8, CR9, CR11, CR12,CR13, CR14, CR15, CR16, CR17, CR18, CR19, CR20, CR21, CR24, CR27, CR28,and CR29 (including all salts, solvates or hydrates thereof).

[0076] The term “pharmaceutically acceptable acid addition salt” as usedherein means any non-toxic organic or inorganic salt of any basecompounds represented by Formulae I, II and/or III or any of theirintermediates. Illustrative inorganic acids which form suitable saltsinclude hydrochloric, hydrobromic, sulfuric and phosphoric acids, aswell as metal salts such as sodium monohydrogen orthophosphate andpotassium hydrogen sulfate. Illustrative organic acids that formsuitable salts include mono-, di-, and tricarboxylic acids such asglycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic,tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic andsalicylic acids, as well as sulfonic acids such as p-toluene sulfonicand methanesulfonic acids. Either the mono or di-acid salts can beformed, and such salts may exist in either a hydrated, solvated orsubstantially anhydrous form. In general, the acid addition salts ofcompounds of Formulae I, II and/or III are more soluble in water andvarious hydrophilic organic solvents, and generally demonstrate highermelting points in comparison to their free base forms. The selection ofthe appropriate salt will be known to one skilled in the art. Othernon-pharmaceutically acceptable salts, e.g. oxalates, may be used, forexample, in the isolation of compounds of Formulae I, II and/or III forlaboratory use, or for subsequent conversion to a pharmaceuticallyacceptable acid addition salt.

[0077] The term “pharmaceutically acceptable basic addition salt” asused herein means any non-toxic organic or inorganic base addition saltof any acid compounds represented by Formulae I, II and/or III or any oftheir intermediates. Illustrative inorganic bases which form suitablesalts include lithium, sodium, potassium, calcium, magnesium or bariumhydroxide. Illustrative organic bases which form suitable salts includealiphatic, alicyclic or aromatic organic amines such as methylamine,trimethylamine and picoline or ammonia. The selection of the appropriatesalt will be known to a person skilled in the art.

[0078] The term “solvate” as used herein means a compound of Formulae I,II and/or III, or a pharmaceutically acceptable salt of a compound ofFormulae I, II and/or III, wherein molecules of a suitable solvent areincorporated in the crystal lattice. A suitable solvent isphysiologically tolerable at the dosage administered. Examples ofsuitable solvents are ethanol, water and the like. When water is thesolvent, the molecule is referred to as a “hydrate”.

[0079] The term an “effective amount” or a “sufficient amount” of anagent as used herein is that amount sufficient to effect beneficial ordesired results, including clinical results, and, as such, an “effectiveamount” depends upon the context in which it is being applied. Forexample, in the context of administering an agent that inhibits cancercell proliferation, an effective amount of an agent is, for example, anamount sufficient to achieve such a reduction in cancer cellproliferation as compared to the response obtained withoutadministration of the agent.

[0080] As used herein, and as well understood in the art, “treatment” isan approach for obtaining beneficial or desired results, includingclinical results. Beneficial or desired clinical results can include,but are not limited to, alleviation or amelioration of one or moresymptoms or conditions, diminishment of extent of disease, stabilized(i.e. not worsening) state of disease, preventing spread of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.

[0081] “Palliating” a disease or disorder means that the extent and/orundesirable clinical manifestations of a disorder or a disease state arelessened and/or time course of the progression is slowed or lengthened,as compared to not treating the disorder.

[0082] The term “modulate” as used herein includes the inhibition orsuppression of a function or activity (such as cell proliferation) aswell as the enhancement of a function or activity.

[0083] To “inhibit” or “suppress” or “reduce” a function or activity,such as cancer cell proliferation, is to reduce the function or activitywhen compared to otherwise same conditions except for a condition orparameter of interest, or alternatively, as compared to anotherconditions.

[0084] The term “animal” as used herein includes all members of theanimal kingdom including human. The animal is preferably a human

[0085] The term “a cell” as used herein includes a plurality of cells.Administering a compound to a cell includes in vivo, ex vivo and invitro treatment.

[0086] The term “cancer cells” as used herein includes all forms ofcancer or neoplastic disease.

[0087] II. Compounds of the Invention

[0088] Novel compounds which are useful in modulating cell proliferationwere prepared. As such the compounds are useful in treating cellproliferative diseases such as cancer.

[0089] Accordingly, the present invention provides compounds of FormulaI, and salts, solvates or hydrates thereof.

[0090] wherein

[0091] R¹ and R² are each independently selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo;

[0092] R³ is selected from the group consisting of H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;

[0093] R⁴ is selected from the group consisting of C(X)R⁵, SO₃Ar, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), P(O)(OH)₂, P(O)(OC₁₋₆alkyl)₂, andC(NH₂)═C(CN)₂;

[0094] X is selected from O,S, NH and N—C₁₋₆alkyl;

[0095] R⁵ is selected from the group consisting of NH₂, OH,NH(CH₂)_(p)Ar, NH(CH₂)_(p)OH, (CH₂)_(p)OC₁₋₆alkyl, C₁₋₆alkyl,C₁₋₆alkoxy, NHNH₂, NHC(O)NH₂, NHC(O)C₁₋₆alkoxy, N-morpholino andN-pyrrolidino; and

[0096] Ar is an aromatic or heteroaromatic group, unsubstituted orsubstituted with 1-4 substituents independently selected from the groupconsisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo;

[0097] n is 0 to 4;

[0098] m is 1 to 4; and

[0099] p is 1-4.

[0100] In embodiments of the invention, compounds of Formula I are thosein which R¹ and R² are each independently selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo. Inpreferred embodiments, R¹ and R² are each independently selected fromthe group consisting of H, OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl,SH, S—C₁₋₄alkyl, O—Si(C₁₋₄alkyl)(C₁₋₄alkyl)(C₁₋₄alkyl), NO₂, CF₃, OCF₃and halo. In more perferred embodiments, R¹ and R² are eachindependently selected from the group consisting of H, OH, OCH₃,O—Si(CH₃)₂(^(t)Bu), S—Me, SH and NO₂. In the most preferred embodimentof the present invention R¹ and R² are both OH or OCH₃ or R¹ is OCH₃ andR² is OH.

[0101] In further embodiments of the present invention, the compounds ofFormula I include those in which R³ is selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar(where n is 0-4). In preferred embodiments, R³ is selected from thegroup consisting of H, OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl,N(C₁₋₄alkyl)(C₁₋₄alkyl), SH, S—C₁₋₄alkyl, NO₂ and halo. In a morepreferred embodiment, R³ is selected from the group consisting of H, OH,OCH₃, SH, SMe, NO₂ and halo. In the most preferred embodment, R³ isselected from the group consisting of H, OH and OCH₃.

[0102] Embodiments of the invention include compounds of Formula Iwherein R⁴ is selected from the group consisting of C(X)R⁵, SO₃Ar, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), P(O)(OH)₂, P(O)(OC₁₋₆alkyl)₂, andC(NH₂)═C(CN)₂ (where m is 1-4). In preferred embodiments, R⁴ is selectedfrom the group consisting of C(X)R⁵ and C(NH₂)═C(CN)₂. More preferably,R⁴ is C(X)R⁵. When R⁴ is C(X)R⁵, embodiments of the invention includecompounds where X is selected from O,S, NH and N—C₁₋₆alkyl and R⁵ isselected from the group consisting of NH₂, OH, NH(CH₂)_(p)Ar,NH(CH₂)_(p)OH, (CH₂)_(p)OC₁₋₆alkyl, C₁₋₆alkyl, C₁₋₆alkoxy, NHNH₂,NHC(O)NH₂, NHC(O)C₁₋₆alkoxy, N-morpholino and N-pyrrolidino (where p is1-4). In preferred embodiments, X is O or S and R⁵ is selected from thegroup consisting of NH₂, OH, NH(CH₂)_(p)Ar, (CH₂)_(p)OH and C₁₋₄alkoxy,(where p is 1-3). Most preferred, are compounds of Formula I wherein Xis O and R⁵ is selected from the group consisting of NH₂, OH,NH(CH₂)_(p)Ar, NH(CH₂)_(p)OH and OCH₃, (where p is 1-2).

[0103] The present invention includes compounds of Formula I wherein theterm “Ar” means an unsubstituted or substituted aryl and/or heteroarylgroup which, in be case of heteroaryl, may contain up to twoheteroatoms, wherein the optional substituents are independentlyselected from the group consisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃and halo, and includes unsubstituted or substituted phenyl, furyl,thienyl, indolyl, naphthyl, quinolyl and the like. In embodiments of thepresent invention, Ar is an unsubstituted phenyl group or a phenyl groupsubstituted with 1-4 substituents optionally selected from the groupconsisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo. Inpreferred embodiments, Ar is an unsubstituted phenyl group or phenylgroup substituted with 1-2 substituents optionally selected from thegroup consisting of OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl,N(C₁₋₄alkyl)(C₁₋₄alkyl), SH, S—C₁₋₄alkyl, NO₂, OF₃, OCF₃ and halo. Inmore preferred embodiments, Ar is an unsubstituted phenyl group orphenyl group substituted with 1-2 substituents optionally selected fromthe group consisting of OH, OCH₃, NH₂, NHCH₃, N(CH₃)_(2,) SH, SCH₃, CF₃,OCF₃ and halo. In the most preferred embodiment, Ar is selected from thegroup consisting of phenyl and 3,4-dihydroxyphenyl.

[0104] The present invention further includes compounds of Formula IIand salts, solvates and hydrates thereof:

[0105] wherein

[0106] R¹ and R² are each independently selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo;

[0107] R³ is selected from the group consisting of H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;

[0108] Ar is an aromatic or heteroaromatic group, unsubstituted orsubstituted with 1-4 substituents, independently selected from the groupconsisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo,

[0109] R⁶ is selected from the group consisting of Ar, OH andOC₁₋₆alkyl;

[0110] X is selected from O and S;

[0111] n is 0-4; and

[0112] p is 1-4.

[0113] In embodiments of the invention, compounds of Formula II arethose in which R¹ and R² are each independently selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo. Inpreferred embodiments, R¹ and R² are each independently selected fromthe group consisting of H, OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl,SH, S—C₁₋₄alkyl, O—Si(C₁₋₄alkyl)(C₁₋₄alkyl)(C₁₋₄alkyl), NO₂, CF₃, OCF₃and halo. In more perferred embodiments, R¹ and R² are eachindependently selected from the group consisting of H, OH, OCH₃,O—Si(CH₃)₂(^(t)Bu), S—Me, SH and NO₂. In the most preferred embodimentof the present invention R¹ and R² are both OH or OCH₃ or R¹ is OCH₃ andR² is OH.

[0114] In further embodiments of the present invention, the compounds ofFormula II include those in which R³ is selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(O₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar(where n is 0-4). In preferred embodiments, R³ is selected from thegroup consisting of H, OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl,N(C₁₋₄alkyl)(C₁₋₄alkyl), SH, S—C₁₋₄alkyl, NO₂ and halo. In a morepreferred embodiment, R³ is selected from the group consisting of H, OH,OCH₃, SH, SMe, NO₂ and halo. In the most preferred embodment, R³ isselected from the group consisting of H, OH and OCH₃.

[0115] The present invention further includes compounds of Formula IIwherein the term “Ar” means an unsubstituted or substituted aryl andheteroaryl group which, in the case of heteroaryl, may contain up to twoheteroatoms, wherein the optional substituents are independentlyselected from the group consisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃and halo, and includes unsubstituted or substituted phenyl, furyl,thienyl, indolyl, naphthyl, quinolyl and the like. In embodiments of thepresent invention, Ar is an unsubstituted phenyl group or a phenyt groupsubstituted with 1-4 substituents optionally selected from the groupconsisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo. Inpreferred embodiments, Ar is an unsubstituted phenyl group or phenylgroup substituted with 1-2 substituents optionally selected from thegroup consisting of OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl,N(CO₁₋₄alkyl)(C₁₋₄alkyl), SH, S—C₁₋₄alkyl, NO₂, CF₃, OCF₃ and halo. Inmore preferred embodiments, Ar is an unsubstituted phenyl group orphenyl group substituted with 1-2 substituents optionally selected fromthe group consisting of OH, OCH₃, NH₂, NHCH₃, N(CH₃)₂, SH, SCH₃, CF₃,OCF₃ and halo. In the most preferred embodiment, Ar is selected from thegroup consisting of phenyl and 3,4-dihydroxyphenyl.

[0116] The compounds of Formula II, include those in which R⁶ isselected from the group consisting of Ar, OH and OC₁₋₆alkyl and p is1-4. In preferred embodiments, R⁶ is selected from the group consistingof Ar and OH and p is 1-2. Most preferably, when R⁶ is Ar, p is 1 andwhen R⁶ is OH, p is 2. Where R⁶ is Ar, Ar means an unsubstituted orsubstituted aryl and/or heteroaryl group which, in the case ofheteroaryl, may contain up to two heteroatoms, wherein the optionalsubstituents are independently selected from the group consisting of OH,C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH,S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo, and includes unsubstituted orsubstituted phenyl, furyl, thienyl, indolyl, naphthyl, quinolyl and thelike. In embodiments of the present invention, Ar is an unsubstitutedphenyl group or a phenyl group substituted with 1-4 substituentsoptionally selected from the group consisting of OH, C₁₋₆alkyl,C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,NO₂, CF₃, OCF₃ and halo. In preferred embodiments, Ar is anunsubstituted phenyl group or phenyl group substituted with 1-2substituents optionally selected from the group consisting of OH,C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl, N(C₁₋₄alkyl)(C₁₋₄alkyl), SH,S—C₁₋₄alkyl, NO₂, CF₃, OCF₃ and halo. In more preferred embodiments, Aris an unsubstituted phenyl group or phenyl group substituted with 1-2substituents optionally selected from the group consisting of OH, OCH₃,NH₂, NHCH₃, N(CH₃)₂, SH, SCH₃, CF₃, OCF₃ and halo. In the most preferredembodiment, Ar is selected from the group consisting of phenyl and3,4-dihydroxyphenyl.

[0117] Compounds of Formula II, further include those in which X isselected from O and S. In preferred embodiments, X is O.

[0118] The present invention also provides a compound of Formula III andsalts, solvates and hydrates thereof:

[0119] wherein

[0120] R¹ and R² are each independently selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo;

[0121] R³ is selected from the group consisting of H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;

[0122] Ar is an aromatic or heteroaromatic group, unsubstituted orsubstituted with 1-4 substituents, independently selected from the groupconsisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo;

[0123] R⁷ is selected from the group consisting of OH, NH₂ andOC₁₋₆alkyl;

[0124] X is selected from O and S, and

[0125] n is 0-4.

[0126] In embodiments of the invention, compounds of Formula III arethose in which R¹ and R² are each independently selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo. Inpreferred embodiments, R¹ and R² are each independently selected fromthe group consisting of H, OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl,SH, S—C₁₋₄alkyl, O—Si(C₁₋₄alkyl)(C₁₋₄alkyl)(C₁₋₄alkyl), NO₂, CF₃, OCF₃and halo. In more perferred embodiments, R¹ and R² are eachindependently selected from the group consisting of H, OH, OCH₃,O—Si(CH₃)₂(^(t)Bu), S—Me, SH and NO₂. In the most preferred embodimentof the present invention R¹ and R² are both OH or OCH₃ or R¹ is OCH₃ andR² is OH.

[0127] In further embodiments of the present invention, the compounds ofFormula III include those in which R³ is selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar(where n is 0-4). In preferred embodiments, R³ is selected from thegroup consisting of H, OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl,N(C₁₋₄alkyl)(C₁₋₄alkyl), SH, S—C₁₋₄alkyl, NO₂ and halo. In a morepreferred embodiment, R³ is selected from the group consisting of H, OH,OCH₃, SH, SMe, NO₂ and halo. In the most preferred embodment, R³ isselected from the group consisting of H, OH and OCH₃.

[0128] The present invention further includes compounds of Formula IIIwherein the term “Ar” means an unsubstituted or substituted aryl and/orheteroaryl group which, in the case of heteroaryl, may contain up to twoheteroatoms, wherein the optional substituents are independentlyselected from the group consisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃and halo, and includes unsubstituted or substituted phenyl, furyl,thienyl, indolyl, naphthyl, quinolyl and the like. In embodiments of thepresent invention, Ar is an unsubstituted phenyl group or a phenyl groupsubstituted with 1-4 substituents optionally selected from the groupconsisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo. Inpreferred embodiments, Ar is an unsubstituted phenyl group or phenylgroup substituted with 1-2 substituents optionally selected from thegroup consisting of OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl,N(C₁₋₄alkyl)(C₁₋₄alkyl), SH, S—C₁₋₄alkyl, NO₂, CF₃, OCF₃ and halo. Inmore preferred embodiments, Ar is an unsubstituted phenyl group orphenyl group substituted with 1-2 substituents optionally selected fromthe group consisting of OH, OCH₃, NH₂, NHCH₃, N(CH₃)₂, SH, SCH₃, CF₃,OCF₃ and halo. In the most preferred embodiment, Ar is selected from thegroup consisting of phenyl and 3,4-dihydroxyphenyl.

[0129] Compounds of Formula III further include those in which R⁷ isselected from the group consisting of OH, NH₂ and OC₁₋₆alkyl. Inpreferred embodiments, R⁷ is selected from the group consisting of OHand NH₂.

[0130] Compounds of Formula III, further include those in which X isselected from O and S. In preferred embodiments, X is O.

[0131] In specific embodiments of the present invention, the compoundsof the invention include:

[0132] (E,E)-2-(benzylamido)-3-styrylacrylonitrile (CR1);

[0133] (E,E)-2-(benzylamido)-3-(3,4-dimethoxystyryl)acrylonitrile (CR2);

[0134](E,E)-2-(benzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR3);

[0135] (E,E)-2-(benzylamido)-3-(3,4dihydroxystyryl)acrylonitdle (CR4);

[0136] (E,E)-2-(phenylethylamido)-3-(3,4-dimethoxystyryl)acrylonitrile(CR5);

[0137](E,E)-2-(phenylethylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile

[0138](E,E)-2-(phenylpropylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR9);

[0139](E,E)-2-(3,4-dihydroxybenzylamido)-3-(3,5imethoxy-4-hydroxystyryl)acrylonitrile(CR11);

[0140](E,E)-2-thioacetamido-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR12);

[0141] (E,E)-2-acetamido-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR13);

[0142] (E,E)-2-carboxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR14);

[0143](E,E)-2-carbomethoxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR15);

[0144] (E,E)-2-acetamido-3-(3,4-dihydroxystyryl)acrylonitrile (CR17);

[0145](E,E)-2-(benzylamido)-3-(3,4bis(t-butyldimethylsilyloxystyryl))acrylonitrile(CR18);

[0146] (E,E)-2-(3,4 dihydroxybenzylamido)-3-styrylacrylonitrile (CR19);

[0147] (E,E)-2-(3,4dihydroxybenzylamido)-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile(CR20);

[0148] (E,E)-2-(3,4dihydroxybenzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile (CR21);

[0149](E,E)-2-(β-ethanolamido)-3-(3,5-dimethoxhydroxystryl)acrylonitrile(CR24);

[0150] (E,E)-2-(benzylamido)-3-(4-nitrostyryl)acrylonitrile (CR27);

[0151](E,E)-2-(3,4dihydroxybenzylamido)-3-(4-nitrostyryl)acrylonitrile(CR28);and

[0152](E,E)-2-(1-amino-2,2-dicyanoethenyl)-3-(4-nitrostyryl)acrylonitrile(CR29).

[0153] In preferred embodiments of the present invention, the compoundsof the invention include:

[0154] (E,E)-2-(benzylamido)-3-styrylacrylonitrile (CR1);

[0155] (E,E)-2-(benzylamido)-3-(3,4-dimethoxystyryl)acrylonitrile (CR2);

[0156](E,E)-2-(benzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR3);

[0157] (E,E)-2-(benzylamido)-3-(3,4dihydroxystyryl)acrylonitrile (CR4);

[0158] (E,E)-2-(phenylethylamido)-3-(3,4-dimethoxystyryl)acrylonitrile(CR5);

[0159](E,E)-2-(phenylpropylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR9);

[0160](E,E)-2-(3,4-dihydroxybenzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR11);

[0161](E,E)-2-thioacetamido-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR12);

[0162] (E,E)-2-acetamido-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR13);

[0163] (E,E)-2carboxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR14);

[0164](E,E)-2-carbomethoxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR15);

[0165] (E,E)-2-acetamido-3-(3,4-dihydroxystyryl)acrylonitrile (CR17);

[0166] (E,E)-2-(3,4 dihydroxybenzylamido)-3-styrylacrylonitrile (CR19);

[0167] (E,E)-2-(3,4dihydroxybenzylamido)-3-(3,4dihydroxysfyryl)acrylonitrite (CR21); and

[0168](E,E)-2-(β-ethanolamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR24).

[0169] In more preferred embodiments of the present invention, thecompounds of the invention include:

[0170] (E,E)-2-(benzylamido)-3-(3,4-dihydroxystyryl)acrylonitrle (CR4);

[0171](E,E)-2-(3,4-dihydroxybenzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR11);

[0172] (E,E)-2-acetamido-3-(3,4dihydroxystyryl)acrylonitrile (CR17);

[0173] (E,E)-2-(3,4 dihydroxybenzylamido)-3-styrylacrylonitrile (CR19);

[0174] (E,E)-2-(3,4dihydroxybenzylamido)-3-(3,4dihydroxystyryl)acrylonitrile (CR21); and

[0175](E,E)-2-(β-ethanolamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR24).

[0176] The present invention includes within its scope, prodrugs of thecompounds of the invention. In general, such prodrugs will be functionalderivatives of a compound of the invention which are readily convertiblein vivo into the compound from which it is notionally derived.Conventional procedures for the selection and preparation of suitableprodrugs are described, for example, in “Design of Prodrugs” ed. H.Bundgaard, Elsevier, 1985.

[0177] Some of the compounds of the invention may have at least oneasymmetric center. Where the compounds according to the invention haveone asymmetric center, the may exist as enantiomers. Where the compoundsof the invention possess two or more asymmetric centers, they mayadditionally exist as diastereomers. It is to be understood that allsuch isomers and mixtures thereof in any proportion are encompassedwithin the scope of the present invention.

[0178] The present invention includes radiolabeled forms of compounds ofthe invention, for example, compounds of the invention labeled byincorporation within the structure ³H or ¹⁴C or a radioactive halogensuch as ¹²⁵I.

[0179] The compounds of the invention may, for example, be derived froman activated cinnamyl compound and an activated cyano-substitutedmethylene compound. A person skilled in the art, therefore, may wish toprovide a generic name for the compounds of the invention based on thecinnamyl moiety. However, generic nomenclature based on the formedacylonitrile moiety, for example, styryl acrylonitrile, would be moreproper.

[0180] III. Methods of Preparing Compounds of the Invention

[0181] In accordance with another aspect of the present invention, thecompounds of the invention can be prepared by processes analogous tothose established in the art. Therefore, compounds of this invention maybe prepared by the reaction sequence shown in Scheme 1:

[0182] Compounds of the general Formulae I, II and/or III useful in thepractice of this invention can be prepared by Knoevenagel condensationof α,β-unsaturated aldehydes, such as cinnamaldehyde or its variousaryl-substituted homologues (IV), with a compound having activeα-methylene group (V) Similar Knoevenagel condensations usingylidenemalononitriles as active α-methylene group components weredescribed in a review (F. Freeman. Chem Rev. 1980, V. 80, P. 329-350).For example, these condensations may be carned out in a polar solvent,such as ethanol, in the presence of catalytic amounts of a weak base,such as β-alanine. Reaction temperatures may be in the range of 20 to100° C., depending on the stability of the materials used in thecondensation.

[0183] Compounds of Formulae IV and/or V may be commercially available,such as cinnamaldehyde, and its 3,5-dimethoxy-4-hydroxy derivative.Other compounds of Formulae IV and/or V may be prepared usingstraightforward procedures. For example, various R¹, R², R³-hydroxysubstituted cinnamaldehydes can be prepared from the correspondingcommercially available aryl substituted cinnamic acids Scheme 2 gives anexample of the preparation of protected 3,4-dihydroxycinnamaldehyde(IVa) starting from 3,4-dihydroxycinnamic acid (VI). At the end of thereaction sequence, the protection groups can be removed using standardmethods well known to those having skill in the art.

[0184] R¹, R², R³ substituents may be also converted from one functionalgroup to another, for example by known reduction of nitro groups intoamino groups and the further transformation into dialkylamino groups, orby known conversion of hydroxy groups to halo groups.

[0185] α-Cyano amides with a reactive methylene group (Va) may beobtained, for example, as described in A. Gazit et.al. J. Med. Chem.,1991, V. 34, P. 1896-1907. For example, by heating methyl cyanoacetate(VII) and an appropriate commercially available amine (VIII) up to 100°C. without presence of a solvent for 12-15 h followed by vacuumdistillation directly from the mixture (for example using a Kugelrohrapparatus), the desired products may be obtained (Scheme 3).

[0186] In some cases the chemistries outlined above may have to bemodified, for instance by use of protective groups, to prevent sidereactions due to reactive groups, such as reactive groups attached assubstituents. This may be achieved by means of conventional protectinggroups, for example as described in “Protective Groups in OrganicChemistry” McOmie, J. F. W. Ed., Plenum Press, 1973 and in Greene, T. W.and Wuts, P. G. M., “Protective Groups in Organic Synthesis”, John Wiley& Sons, 1991.

[0187] The formation of a desired compound salt is achieved usingstandard techniques. For example, the neutral compound is treated withan acid or base in a suitable solvent and the formed salt is isolated byfiltration, extraction or any other suitable method.

[0188] The formation of solvates of the compounds of the invention willvary depending on the compound and the solvate. In general, solvates areformed by dissolving the compound in the appropriate solvent andisolating the solvate by cooling or using an antisolvent. The solvate istypically dried or azeotroped under ambient conditions.

[0189] Prodrugs of the compounds of the invention may be conventionalesters formed with available hydroxy, amino or carboxyl group. Forexample, when R¹, R² or R³ is OH in a compound of Formulae I, II and/orIII, it may be acylated using an activated acid in the presence of abase, and optionally, in inert solvent (e.g. an acid chloride inpyridine). Some common esters which have been utilized as prodrugs arephenyl esters, aliphatic (C₈-C₂₄) esters, acyloxymethyl esters,carbamates and amino acid esters.

[0190] A radiolabeled compound of the invention may be prepared usingstandard methods known in the art. For example, tritium may beincorporated into a compound of the invention using standard techniques,for example by hydrogenation of a suitable precursor to a compound ofthe invention using tritium gas and a catalyst. Alternatively, acompound of the invention containing radioactive iodo may be preparedfrom the corresponding trialkyltin (suitably trimethyltin) derivativeusing standard iodination conditions, such as [¹²⁵I] sodium iodide inthe presence of chloramine-T in a suitable solvent, such asdimethylformamide. The trialkyltin compound may be prepared from thecorresponding non-radioactive halo, suitably iodo, compound usingstandard palladium-catalyzed stannylation conditions, for examplehexamethyiditin in the presence of tetrakis(triphenylphosphine)palladium (0) in an inert solvent, such as dioxane, and at elevatedtemperatures, suitably 50-100° C.

[0191] IV. Uses

[0192] As hereinbefore mentioned, the inventors have prepared novelcompounds of the Formulae I, II and III. Accordingly, the presentinvention includes all uses of the compounds of the invention includingtheir use in therapeutic methods and compositions for modulating cellproliferation, their use in diagnostic assays and their use as researchtools.

[0193] In one aspect, the present invention provides a method formodulating cell proliferation comprising administering an effectiveamount of a compound of the invention to a cell or animal in needthereof. Preferably, the invention provides a method of inhibiting cellproliferation comprising administering an effective amount of a compoundof the invention to a cell or animal in need thereof. In particular, themethod of the invention is useful in inhibiting the proliferation ofabnormal but not normal cells. Abnormal cells include any type of cellthat is causative of or involved in a disease or condition and whereinit is desirable to modulate or inhibit the proliferation of the abnormalcell to treat the disease or condition. Examples of abnormal cellsinclude malignant or cancerous cells as well as cell thatover-proliferate in inflammatory conditions.

[0194] It has been determined that some of the compounds of theinvention are very effective at killing cancer cells while at the sametime they do not kill normal cells. These properties make the compoundsof the invention extremely useful as anti-cancer agents. Accordingly, inone embodiment, the present invention provides a method of inhibitingthe proliferation of a cancer cell comprising administering an effectiveamount of a compound of the invention to a cell or animal in needthereof.

[0195] The cancer cell that can be treated with a compound of theinvention may be any type of cancer including, but not limited to,hematopoietic malignancies, including leukemias, lymphomas, and myelomasas well as other types of cancer including sarcomas, carcinomas,melanomas, adenomas, nervous system cancers and genitourinary cancers.Examples of leukemias include acute lymphoblastic leukemia (ALL), acutemyelocytic leukemia (AML), chronic myeloid leukemia (CML), chroniclymphocytic leukemia (CLL) and juvenile myelo-monocytic leukemia (JMML).The types of ALL that may be treated with the compounds of the inventioninclude cells that express a bcr-abl fusion protein, such asPhiladelphia positive ALL cells, as well as Philadelphia negative ALLcells. Examples of lymphomas include B-cell Burkitt's lymphoma,Hodgkin's lymphomas, non-Hodgkin's lymphomas, including the Ki-1positive anaplastic large cell lymphomas, T cell lymphomas and rarelymphomas such as the histiocytic lymphomas. Examples of myelomasinclude multiple myelomas.

[0196] In a specific embodiment, the present invention provides a methodof inhibiting the proliferation of a cancer cell comprisingadministering an effective amount of a compound selected from the groupof compounds:

[0197] (E,E)-2-(benzylamido)-3-styrylacrylonitrile (CR1);

[0198] (E,E)-2-(benzylamido)-3-(3,4-dimethoxystyryl)acrylonitrile (CR2);

[0199](E,E)-2-(benzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR3)

[0200] (E,E)-2-(benzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile (CR4);

[0201] (E,E)-2-(phenylethylamido)-3-(3,4-dimethoxystyryl)acrylonitrile(CR5);

[0202](E,E)-2-(phenylethylamido)-3-(3,6-dimethoxy-4-hydroxystyryl)acrylonitrite(CR8);

[0203](E,E)-2-(phenylpropylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR9);

[0204] (E,E)-2-(3,4-dihydroxybenzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile (CR11);

[0205](E,E)-2-thioacetamido-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR12);

[0206] (E,E)-2-acetamido-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR13);

[0207] (E,E)-2-carboxy-3-(3,5-dimethoxyhydroxystyryl)acrylonitrile(CR14);

[0208](E,E)-2-carbomethoxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR15);

[0209](E,E)-2-acetamido-3-[3,4-bis(t-butyidimethylsilyloxystyryl)]aorylonitrile(CR16);

[0210] (E,E)-2-acetamido-3-(3,4dihydroxystyryl)acrylonitrile (CR17);

[0211](E,E)-2-(benzylamido)-3-(3,4-bis(t-butyldimethylsilytoxystyryt))acrylonitrile(CR18);

[0212] (E,E)-2-(3,4 dihydroxybenzylamido)-3-styrylacrylonitrile (CR19),

[0213] (E,E)-2-(3,4dihydroxybenzylamido)-3-[3,4-bis(t-butyidimethylsilyloxystyryl)]acrylonitrile(CR20);

[0214] (E,E)-2-(3,4dihydroxybenzylamido)-3-(3,4dihydroxystyryl)acrylonitrile (CR21);

[0215](E,E)-2-(β-ethanolamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR24);

[0216] (E,E)-2-(benzylamido)-3-(4-nitrostyryl)acrylonitrile (CR27);

[0217](E,E)-2-(3,4dihydroxybenzylamido)-3-(4-nitrostyryl)acrylonitrile(CR28);and

[0218](E,E)-2-(1-amino-2,2-dicyanoethenyl)-3-(4-nitrostyryl)acrylonitrile(CR29).

[0219] In a preferred embodiment, the present invention provides amethod of inhibiting the proliferation of a cancer cell comprisingadministering an effective amount of a compound selected from the groupof compounds:

[0220] (E,E)-2-(benzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile (CR4);

[0221](E,E)-2-(3,4-dihydroxybenzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR11);

[0222] (E,E)-2-acetamido-3-(3,4-dihydroxystyryl)acrylonitrile (CR17);

[0223] (E,E)-2-(3,4 dihydroxybenzylamido)-3-styrylacrylonitrile (CR19);

[0224] (E,E)-2-(3,4dihydroxybenzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile (CR21); and

[0225](E,E)-2-(β-ethanolamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR24).

[0226] One skilled in the art can determine which compounds of theinvention would have therapeutic utility, for example, in inhibitingcell proliferation in any type of cancer or cell proliferative disorder.Compounds may be examined for their efficacy in inhibiting cell growthin cell proliferation assays such as those described herein in Examples35-56 Accordingly, the methods, uses and compositions of the inventionare meant to include only those compounds having the desired effect.

[0227] The ability of the compounds of the invention to inhibit thegrowth of cancer cells, in particular hematopoetic cell malignancies, invitro and in vivo was examined. Several of the compounds tested werefound to eliminate cancerous cell growth in culture at sub-micromolardoses. In particular, CR4, CR11 and CR19 were found to be highlyeffective against a variety of cell types, such as Acute LymphoblasticLeukemia, Philadelphia positive Leukemia and Acute Myeloid Leukemia. Lownanomolar doses of both CR4 and CR19 were highly toxic to cancer cells,while normal cell growth and differentiation were unaffected. Theseeffects were obtained by long term exposure to low levels of thecompounds. Accordingly, in one aspect, this invention provides a methodof inhibiting the proliferation of a hematopoietic cancer cell byadministering an effective amount of a compound of the invention,preferably, CR4 or CR11 or CR19, to a cell or animal in need thereof.

[0228] It has been determined that the compound CR4 is capable ofeffectively killing human Philadelphia positive acute lymphoblasticleukemia cells in vivo, using a murine model. CR4 efficiently reducedtumor load and infiltration of the organs by the ALL cells. The dosesrequired to eliminate cancer cell growth do not result in detectablenon-specific damage to the animal.

[0229] It has also been determined that the compounds of the invention,such as CR4 and CR11, are effective as ex vivo purging agents. For exvivo administration, bone marrow cells may be removed from a patientwith cancer and purged ex vivo with a compound of the invention. Such apurging will kill the tumor cells while leaving the normal bone marrowcells intact. After purging, the cells can be washed and reintroducedinto the patient.

[0230] During ex vivo purging assays the cells were exposed torelatively high doses of the compounds (50 μM-100 μM) for short (1-24hours) periods of time, resulting in the elimination of cancer cellgrowth, while normal bone marrow cells exposed to the same doses overthe same period of time were relatively unaffected. Cancer cell deathwas effected by the induction of apoptosis. Accordingly, in anotheraspect of the invention, there is provided a method for killing cancercells by ex vivo treatment of bone marrow from a patient with cancerwith a compound of the invention, preferably CR4 and CR11 and thenre-introducing the treated (or purged) bone marrow into the patient.

[0231] In addition to cancer, the compounds of the invention are usefulin treating other conditions involving aberrant or abnormal cellproliferation. Other cell proliferative disorders that may be treated bythe present invention include inflammatory diseases, allergies,autoimmune disease, graft rejection psoriasis, restenosis,artherosclerosis, and any other disorder wherein it is desirable toinhibit, prevent or suppress cell growth. Compounds of the invention maybe tested for their efficacy in a particular cell proliferation disorderusing assays and techniques known to those of skill in the art. Forexample, the following references provide assays for various conditions.Rheumatoid Arthritis: “Regulation of IL-15-Simulated TNF-alphaProduction by Rolipram”, Journal of Immunology (1999) volume 163 page8236 by C. S. Kasyapa et al. Allergy: “A novel Lyn-Binding PeptideInhibitor Blocks Eosinophil Differentiation, Survival, and Airwayeosinophilic inflammation”. Journal of Immunology (1999) volume 163 page939 by T. Adachi et al. Psoriasis: Journal of Immunology (2000) volume165 page 224 “Inhibition of Keratinocyte apoptosis by IL-15: a newparamete in the pathegenosis of psoriasis” by R. Uchert (there is anumiatt over the U). Psoriasis: International Archives of allergy andImmunology (2000) Volume 123 page 275. “T-cell receptor mimic peptidesand their potential application in T-cell mediated disease” by A. H.Enk.

[0232] The compounds of the invention are tyrosine kinase modulators andare useful in modulating tyrosine kinase activity, including theinhibition of tyrosine kinase activity, for the treatment of variousconditions such as all proliferative disorders as mentioned above,Accordingly, the invention provides a method of modulating tyrosinekinase activity by administering an effective amount of a compound ofthe invention to a cell or animal in need thereof. In a further aspect,the invention provides a method of inhibiting tyrosine kinase activityby administering an effective amount of a compound of the invention to acell or animal in need thereof.

[0233] While the compounds of the invention may act by inhibitingtyrosine kinase activity, one of skill in the art will appreciate thatother modes or mechanisms of action for the compounds of the inventionare possible.

[0234] The compounds of the invention are preferably formulated intopharmaceutical compositions for administration to human subjects in abiologically compatible form suitable for administration in vivo.Accordingly, in another aspect, the present invention provides apharmaceutical composition comprising a compound of the invention inadmixture with a suitable diluent or carrier.

[0235] The compositions containing the compounds of the invention can beprepared by known methods for the preparation of pharmaceuticallyacceptable compositions which can be administered to subjects, such thatan effective quantity of the active substance is combined in a mixturewith a pharmaceutically acceptable vehicle. Suitable vehicles aredescribed, for example, in Remington's Pharmaceutical Sciences(Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., USA 1985). On this basis, the compositions include, albeit notexclusively, solutions of the substances in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids.

[0236] The compounds of this invention may be used in the form of thefree base, in the form of salts, solvates and as hydrates. All forms arewithin the scope of the invention. Acid addition salts may be formed andprovide a more convenient form for use; in practice, use of the saltform inherently amounts to use of the base form. The acids which can beused to prepare the acid addition salts include preferably those whichproduce, when combined with the free base, pharmaceutically acceptablesalts, that is, salts whose anions are non-toxic to the animal organismin pharmaceutical doses of the salts, so that the beneficial propertiesinherent in the free base are not vitiated by side effects ascribable tothe anions. Although pharmaceutically acceptable salts of the basiccompounds are preferred, all acid addition salts are useful as sourcesof the free base form even if the particular salt per se is desired onlyas an intermediate product as, for example, when the salt is formed onlyfor the purposes of purification and identification, or when it is usedas an intermediate in preparing a pharmaceutically acceptable salt byion exchange procedures.

[0237] Pharmaceutically acceptable salts within the scope of theinvention include those derived from the following acids; mineral acidssuch as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamicacid; and organic acids such as acetic acid, citric acid, lactic acid,tartaric acid, malonic acid, methanesuffonic acid, ethanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid,quinic acid, and the like.

[0238] In accordance with the methods of the invention, the describedcompounds or salts or solvates thereof may be administered to a patientin a variety of forms depending on the selected route of administration,as will be understood by those skilled in the art. The compositions ofthe invention may be administered orally or parenterally. Parenteraladministration includes intravenous, intraperitoneal, subcutaneous,intramuscular, transepithelial, nasal, intrapulmonary, intrathecal,rectal and topical modes of administration. Parenteral administrationmay be by continuous infusion over a selected period of time.

[0239] A compound of the invention or a salt or solvate thereof may beorally administered, for example, with an inert diluent or with anassimilable edible carder, or it may be enclosed in hard or soft shellgelatin capsules, or it may be compressed into tablets, or it may beincorporated directly with the food of the diet. For oral therapeuticadministration, the compound of the invention may be incorporated withexcipient and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0240] A compound of the invention may also be administered parenterallyor intraperitoneally. Solutions of a compound of the invention as a freebase or pharmacologically acceptable salt or solvate can be prepared inwater suitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, DMSO and mixtures thereof with or without alcohol, and in oils.Other solvents include taxol formulation, CMC, Tween20. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. A person skilled in the artwould know how to prepare suitable formulations. Conventional proceduresand ingredients for the selection and preparation of suitableformulations are described, for example, in Remington's PharmaceuticalSciences (1990—18th edition) and in The United States Pharmacopeia: TheNational Formulary (USP 24 NF₁₉) published in 1999.

[0241] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersion and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists.

[0242] The compounds of the invention may be administered to an animalalone or in combination with pharmaceutically acceptable carriers, asnoted above, the proportion of which is determined by the solubility andchemical nature of the compound, chosen route of administration andstandard pharmaceutical practice.

[0243] The dosage of the compounds and/or compositions of the inventioncan vary depending on many factors such as the pharmacodynamicproperties of the compound, the mode of administration, the age, healthand weight of the recipient, the nature and extent of the symptoms, thefrequency of the treatment and the type of concurrent treatment, if any,and the clearance rate of the compound in the animal to be treated. Oneof skill in the art can determine the appropriate dosage based on theabove factors. The compounds of the invention may be administeredinitially in a suitable dosage that may be adjusted as required,depending on the clinical response. As an example, the compounds of theinvention can be administered in a range from about 1 nanomolar to about100 micromolar, preferably 50 nanomolar to 50 micromolar. For ex vivotreatment of cells over a short period, for example for 30 minutes to 1hour or longer, higher doses of compound may be used than for long termin vivo therapy; for example, concentrations of 5 μM or higher may beused.

[0244] The present invention also includes a use of a compound orcomposition of the invention in order to inhibit cell proliferation,preferably cancer cell proliferation. The present invention furtherincludes a use of a compound or a composition of the invention toprepare a medicament to inhibit cell proliferation, preferably cancercell proliferation.

[0245] The compounds of the invention can be used alone or incombination with other agents that modulate tyrosine kinase activity orin combination with other types of treatment (which may or may notmodulate tyrosine kinase activity) for cell proliferative disorders.Agents known in the art that inhibit tyrosine kinase activity include,but are not limited to, antisense nucleic acid and ribozymes targeted tonucleic acid encoding a receptor tyrosine kinase, antibodies able tomodulate tyrosine kinase activity and other small molecule tyrosinekinase inhibitors such as those described in U.S. Pat. Nos. 5,891,917,5,217,999, 5,773,476, 5,935,993, 5,656,655, 5,677,329 and 5,789,427.There are various examples of other types of treatment for cellproliferative disorders currently used to treat different types ofcancers. The general treatments are based on the cancer type and do notspecifically target tyrosine kinase activity. In a particular aspect ofthe present invention, the compounds of the invention may be used incombination with other therapies and therapeutics to treat leukemia.

[0246] In addition to the abovementioned therapeutic uses, the compoundsof the invention are also useful in diagnostic assays, screening assaysand as research tools.

[0247] In diagnostic assays the compounds of the invention may be usefulin identifying or detecting a cell proliferative disorder. In such anembodiment, the compounds of the invention may be radiolabelled (ashereinbefore described) and contacted with a population of cells. Thepresence of the radiolabelled on the cells may indicate a cellproliferative disorder. In a specific embodiment, the radiolabelledcompounds of the invention may be used to detect the presence of cellsexpressing a bcr-abl fusion protein.

[0248] In screening assays, the compounds of the invention may be usedto identify other compounds that modulate cell proliferation or tyrosinekinase activity. As research tools, the compounds of the invention maybe used in receptor binding assays and assays to study the localizationof tyrosine kinases. In such assays, the compounds may also beradiolabelled.

[0249] Novel compounds and methods for modulating cell proliferationalso are described in PCT/CA 01/______, filed Apr. 12, 2001, whichclaims the priority benefit of U.S. Provisional Application Serial No.60/196,936, filed Apr. 13, 2000, the disclosures of which areincorporated herein by reference in their entirety.

[0250] The following non-limiting examples are illustrative of thepresent invention:

EXAMPLES

[0251] Materials and Methods For Examples 1-34

[0252]¹H NMR spectra were obtained on a Varian Unity Plus spectrometer(USA) at 500 MHz with tetramethylsilane (TMS, Me₄Si) as an internalstandard (δ=0). Electrospray mass spectra were recorded on an API IIIPlus triple quadrupole mass spectrometer (USA), with a directintroduction of the samples into the ionization source. Thin layerchromatography was performed with UV-254 aluminum-backed TLC sheets of0.25 mm thickness (Kieselgel 60 F₂₅₄, Merck, Germany) HPLC separation ofthe compound of Example 13 was performed on a Waters 600 chromatograph(USA), column Nova-Pak C18 3.9×300 mm (Waters, USA). Vacuumdistillations were done using Kugelrohr apparatus (Aldrich, USA) atstated temperatures of an oven. 3,5-Dimethoxy-4-hydroxycinnamaldehyde,4-nitrocinnamaldehyde, 3,4-dimethoxycinnamic acid, 3,4-dihydroxycinnamicacid, 3,4-dimethoxybenzylamine, benzylamine, phenylethylamine,phenylpropylamine, methyl cyanoacetate, 2-cyanothioacetamide,2-cyanoacetamide, cyanoacetic acid, β-ethanolamine,2-amino-1-propene-1,1,3-tricarbonitrile were purchased from Aldrich(USA) and were used as received. The reagents were from Aldrich (USA).Solvents were purchased from Caledon (Canada).

Example 1 N-(Cyanoacetyl)3,4-dimethoxybenzylamide (A₁)

[0253]

[0254] To 3,4-dimethoxybenzylamine (2.7 ml, 18 mmol) methyl cyanoacetatewas added (1.6 ml, 18 mmol). The reaction was heated for 14 h at 100° C.Cooling gave a dark brown solid which was recrystallized from ethanol togive 2.90 g of the product (69% yield).

[0255] The product gave the following analytical data:

[0256] NMR (CD₃COCD₃, δ, ppm): 3.62 (s, 2H, CH₂CN), 3.78 (s, 6H,(OMe)₂), 4.34 (brs, 2H, NHCH₂Ph), 6.84 (dd, 1H, J 1.95 and 8.1 Hz, H⁶),6.88 (d, 1H, J 8.1 Hz, H⁵), 6.93 (d, 1H, J 1.95 Hz, H²), 7.80 (br.s.,1H, NH).

[0257] MS, m/e (rel. intensity, %): 235 (19) [M+H]⁺, 252 (100) [M+NH₄]⁺,257 (33) [M+Na]⁺.

Example 2 N-(Cyanoacetyl)3,4-dihydroxybenzylamide (A₂)

[0258]

[0259] To N-(cyanoacetyl)3,4-dimethoxybenzylamide (Example 1, 0.2 g,0.85 mmol) in 20 ml of CH₂Cl₂ boron tribromide was added under argon at−78° C. (0.24 ml, 2.56 mmol) in 2.5 ml of CH₂Cl₂. After 2 h the reactionwas brought to room temperature and stirred overnight. The reaction wascooled to 0° C., 10 ml of 1N HCl was added, the solution was extractedwith 3×50 ml of ethyl acetate, the organic phase was washed to neutralpH, dried with MgSO₄, and taken to dryness. The residue was purified bysilica gel chromatography (CHCl₃-MeOH, 20:1) to give a yellow solid(0.07 g, 40% yield). The product gave the following analytical data;

[0260] NMR (CD₃COCD₃, δ, ppm): 2.83 (s, (OH)₂), 3.60 (s, 2H, CH₂CN),4.25 (br.s., 2H, NHCH₂Ph), 6.63 (dd, 1H, J 1.95 and 8.1 Hz, H⁶), 6.75(d, 1H, J 8.1 Hz, H⁵), 6.79 (d, 1H, J 1.95 Hz, H²), 7.71 (br.s., 1H,NH).

[0261] MS, m/e (rel. intensity, %): 207 (38) [M+H]⁺, 224 (100) [M+NH₄]⁺,229 (2.6) [M+Na]⁺.

Example 3(E,E)-2-(3,4-Dihydroxybenzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR11)

[0262]

[0263] To 3,5-dimethoxy-4-hydroxycinnamaldehyde (0.042 g, 0.2 mmol) andN-(cyanoacetyl)3,4-dihydroxybenzylamide (Example 2, 0.042 g, 0.2 mmol)in 10 ml of ethanol 3 mg of β-alanine was added and the reaction wasrefluxed for 6 h. Water was added and the solid was recrystallized from5 ml of ethanol twice to give 0.06 g (75%) of a red solid. The productgave the following analytical data:

[0264] NMR (CD₃COCD₃, δ, ppm): 2.81 (s, (OH)₃), 3.89 (s, 6H, (OMe)₂),4.39 (br.s., 2H, NHCH₂Ph), 6.68 (dd, 1H, J 1.95 and 8.1 Hz, H⁶′), 6.76(d, 1H, J 8.1 Hz, H⁵′), 6.86 (d, 1H, J 1.95 Hz, H²′), 7.07 (br.s, 2H,H²⁺⁶), 7.16 (dd, 1H, J 11.7 and 15.1 Hz, PhCCHCCN olefinic), 7.37 (d,1H, J 15.1 Hz, PhCH olefinic), 7.70 (br.s, 1H, NH), 7.98 (dd, 1H, J 0.75and 11.7 Hz, CHCN olefinic).

[0265] MS, m/e (rel. intensity, %): 397 (100) [M+H]⁺, 414 (14) [M+NH₄]⁺.

Example 4 N-(Cyanoacetyl)benzylamide (A₃)

[0266]

[0267] The compound was prepared as described in Example 1 by addingmethyl cyanoacetate (1.3 ml, 14 mmol) to benzylamine (1.5 ml, 14 mmol).The compound was distilled in vacuo directly from the reaction mixture(Kugelrohr apparatus (Aldrich), 0.1 mm Hg, T. oven 180-190° C.) to givean off-white solid (2.34 g, 95%). The product gave the followinganalytical data,

[0268] NMR (CD₃COCD₃, δ, ppm): 3.39 (s, 2H, CNCH₂), 4.46 (d, 2H, J 5.4Hz, NHCH₂Ph), 6.40 (br.s., 1H, NH), 7.24-7.36 (m, 5H, Ph).

[0269] MS, m/e (rel. intensity, %): 175 (64) [M+H]⁺, 192 [M+NH₄]⁺.

Example 5 3,4-Dimethoxycinnamyl alcohol (A₆)

[0270]

[0271] To a solution of 0.42 g (2.0 mmol) of 3,4-dimethoxycinnamic acidin 50 ml MeOH was added SOCl₂ (50 μl) and the mixture was stirred at 60°C. for 5 h. Methanol was taken to dryness and the obtained3,4-dimethoxycinnamic acid methyl ester was reduced with 1M THF solutionof diisobutylaluminum hydride (8.0 mmol) in absolute THF (50 ml) at 20°C. for 1 h. Water was added, the mixture was extracted with EtOAc, driedwith MgSO₄ and distilled in vacuo (Kugelrohr apparatus (Aldrich), 0.1 mmHg, T. oven 185-190° C.) giving an off-white solid, yield 0.36 g (92%),m.p. 70-71° C. The product gave the following analytical data:

[0272] NMR (CD₃COCCD₃, δ, ppm): 3.77, 3.82 (2×s, 2×3H, OMe+OMe), 4.19(d, 2H, J 5.0 Hz, CH₂OH), 6.25 (dt, 1H, J 5.0 and 15.5 Hz, PhCCHolefinic), 6.51 (d, 1H, J 15.5 Hz, PhCH olefinic), 6.89 (m, 2H, HS⁵⁺⁶),7.05 (br.s., 1H, H²).

[0273] MS, m/e (rel. intensity, %): 177 (100) [M−OH]+, 195 (4) [M+H]⁺,212 (59) [M+NH₄]⁺, 217 (26) [M+Na]⁺.

Example 6 3,4-Dimethoxycinnamaldehyde (A₇)

[0274]

[0275] To a mixture of pyridinium dichromate (3.88 g, 10.3 mmol) and 4 gof finely grounded freshly activated molecular sieves 3A in 20 ml ofCH₂Cl₂ 3,4-dimethoxycinnamyl alcohol in 10 ml of CH₂Cl₂ (Example 5, 1.00g, 5.1 mmol) was added The reaction was stirred for 2 h, 0.5 ml ofmethanol was added, the residue was passed through silica gel and washedwith 300 ml of ethyl acetate. After evaporation the compound waspurified by silica gel chromatography (hexane-EtOAc, 5:1) leading to acrystallizing oil (0.62 g, 63%).

[0276] The product gave the following analytical data:

[0277] NMR (CD₃COCD₃, δ, ppm): 3.90 (2×s, 2×3H, OCH₃+OCH₃), 6.70 (dd,1H, J 7.6 and 16.0 Hz, PhC═CH olefinic), 7.05 (d, 1H, J 8.3 Hz, H⁵),7.28 (dd, 1H, J 1.4 and 8.3 Hz, H⁶), 7.37 (d, 1H, J 1.4 Hz, H²), 7.60(d, 1H, J 16.0 Hz, PhCH olefinic), 9.65 (d, 1H, J 7.6 Hz, CHO).

[0278] MS, m/e (rel. intensity, %): 193 (100) [M+H]⁺, 210 (26) [M+NH₄]⁺.

Example 7 (E,E)-2-(Benzylamido)-3-(3,4-dimethoxystyryl)acrylonitrile(CR2)

[0279]

[0280] The compound was prepared as described in Example 3, by adding3,4-dimethoxycinnamaldehyde (Example 6, 0.04 g, 0.2 mmol) toN-(cyanoacetyl)benzylamide (Example 4, 0.036 g, 0.2 mmol). Afterrefluxing for 1 h and recrystallization from ethanol a yellow solid wasobtained (0.045 g, 62%). The product gave the following analytical data:

[0281] NMR (CD₃COCD₃, δ, ppm): 3.90 (s, 2×3H, OMe+OMe), 4.57 (d, 2H, J<2Hz, NHCH₂Ph), 7.08 (br.s., 1H, H²), 7.17 (dd, 1H, J 11.5 and 15.2 Hz,PhCCHCCN olefinic), 7.23-7.42 (m, 8H, aromatic+H⁵+H⁶+PhCH olefinic),7.90 (br.t, 1H, NH), 8.05 (dd, 1H, J 0.55 and 11.5 Hz, CHCN olefinic).

Example 8 (E,E)-2-(Benzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile(CR4)—Method A

[0282]

[0283] Boron tribromide (0.033 ml, 0.34 mmol) was added to(E,E)-2-(benzylamido)-3-(3,4-dimethoxystyryl)acrylonitrile (Example 7,0.04 g, 0.11 mmol). The residue was purified by silica gelchromatography (CHCl₃-MeOH, 10:1) to give an orange solid (0.02 g, 55%yield). The product gave the following analytical data:

[0284] NMR (CD₃COCD₃, δ, ppm): 2.86 (br.s., 2H, (OH)₂), 4.55 (m, 2H,NHCH₂Ph), 6.90-7.42 (m, 10H, Ph+Ph′+olefinic), 7.87 (br.s., 1H, NH),8.02 (dd, 1H, J<0.5 and 11.4 Hz, CHCN olefinic).

[0285] MS, m/e (rel. intensity, %): 295 (61) [M+H−CN]⁺, 321 (100)[M+H]⁺, 338 (30) [M+NH₄]⁺.

Example 9 Methyl ester of 3,4-bis(t-butyldimethylsilyloxy)cinnamic acid(A₈)

[0286]

[0287] To a solution of 3.6 g (20 mmol) of 3,4-dihydroxycinnamic acid in300 ml MeOH was added SOCl₂ (100 μl) and the mixture was stirred at 60°C. for 5 h. Methanol was taken to dryness and the obtained methyl esterwas treated up with 10.2 g (68 mmol) of t-BuMe₂SiCl and 9.2 g (136 mmol)of imidazole in 100 ml DMF at 50° C. for 0.5 h. Mixture was diluted withwater and extracted with hexane. Hexane was taken to dryness. Theresidue was distilled in vacuo (Kugelrohr apparatus (Aldrich), 0.1 mmHg, T. oven 200-210° C.) and crystallized from hexane at −20° C. givinga white solid, yield 7.5 g (89%), m.p. 57-58° C. The product gave thefollowing analytical data:

[0288] MS, m/e (rel. intensity, %): 423 (100) [M+H]⁺, 440 (98) [M+NH₄]⁺.

Example 10 3,4-Bis(t-butyldimethylsilyloxy)cinnamyl alcohol (A₉)

[0289]

[0290] The compound was prepared as described in Example 5 by treatingof 3,4-dihydroxycinnamic acid bis(BDMS) ether methyl ester (Example 9,0.42 g, 1.0 mmol) with 1M THF solution of diisobutylaluminum hydride(4.0 mmol) in absolute THF (25 ml) at 20° C. for 1 h. After distillingin vacuo (Kugelrohr apparatus (Aldrich), 0.1 mm Hg, T. oven 185-200° C.)a white viscous oil was obtained, yield 0.33 g (85%). The product gavethe following analytical data:

[0291] NMR (CD₃COCD₃, δ, ppm): 0.23, 0.24 (2×s, 2×6H, Me₂Si+Me₂Si),1.00, 1.02 (2×s, 2×9H, t-BuSi+t-BuSi), 4.19 (d, 2H, J 4.9 Hz, CH₂OH),6.22 (dt, 1H, J 4.9 and 16.0 Hz, PhCCH olefinic), 6.49 (d, 1H, J 16.0Hz, PhCH olefinic), 6.85 (d, 1H, J 8.2 Hz, H⁵), 6.92 (dd, 1H, J 2.1 and8.2 Hz, H⁶), 6.97 (d, 1H, J 2.1 Hz, H²).

[0292] MS, m/e (rel. intensity, %): 377 (100) [M—OH]⁺, 395 (2) [M+H]⁺,412 (15) [M+NH₄]⁺.

Example 11 3,4-Bis(t-butyldimethylsilyfoxy)cinnamaldehyde (A₁₀)

[0293]

[0294] The compound was prepared as described in Example 6 by adding3,4-bis(t-butyldimethylsilyloxy)cinnamyl alcohol (Example 10, 0.2 g, 0.5mmol) in 5 ml of CH₂Cl₂ to a mixture of pyridinium dichromate (0.38 g, 1mmol) and 1 g molecular sieves 3 Å in 20 ml of CH₂Cl₂ The residue waspassed through silica gel and washed with 300 ml of EtOAc-hexane, 1:1.After evaporation the compound was purified by silica gel chromatography(hexane-EtOAc, 5:1) leading to an oil (0.15 g, 76%) The product gave thefollowing analytical data:

[0295] NMR (CD₃COCD₃, δ, ppm): 0.26 and 0.28 (2×s, 2×6H, Me₂Si+Me₂Si),1.01 and 1.02 (2×s, 2×9H, t-BuSi+t-BuSi), 6.60 (dd, 1H, J 7.7 and 15.9Hz, PhCCH olefinic), 7.01 (dd, 1H, J<0.5 and 8.9 Hz, H⁶), 7.27 (m, 2H,H²⁺⁵), 7.60 (d, 1H, J 15.9 Hz, PhCH olefinic), 9.65 (d, 1H, d 7.7 Hz,CHO).

[0296] MS, m/e (rel. intensity, %): 367 (3) [M+H−CN]⁺, 393 (100) [M+H]⁺,410 (10) [M+NH₄]⁺.

Example 12(E,E)-2-(Benzylamido)-3-(3,4-bis(t-butyldimethylsilyloxystyryl))acrylonitrile(CR18)

[0297]

[0298] The compound was prepared as described in Example 3 by adding3,4-bis(t-butyldimethylsilyloxy)cinnamaldehyde (Example 11, 0.1 00 g,0.26 mmol) to N-(cyanoacetyl)benzylamide (Example 4, 0.044 g, 0.26 mmol.After refluxing for 2.5 h purification by silica gel chromatography(hexane-EtOAc, 15:1) provided a yellow solid (0.090 g, 64%). The productgave the following analytical data:

[0299] NMR (CD₃COCD₃, δ, ppm): 0.24 and 0.25 (2×s, 2×6H, Me₂Si+Me₂Si),1.01 and 1.02 (2×s, 2×9H, t-BuSi+t-BuSi), 4.55 (br.s., 2H, NHCH₂Ph),7.00 (d, 1H, J 8.5 Hz, H⁴), 7.12 (dd, 1H, J 11.7 and 15.6 Hz, PhCCHCCNolefinic), 7.24-7.43 (m, 8H, aromatic and olefinic protons), 7.93(br.s., 1H, NH), 8.02 (dd, 1H, J<0.5 and 11.7 Hz, CHCN olefinic).

[0300] MS, m/e (rel. intensity, %): 523 (30) [M+H−CN]⁺, 540 (24)[M+NH₄−CN]⁺, 549 (89) [M+H]⁺, 566 (100) [M+NH₄]⁺.

Example 13 (E,E)-2-(Benzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile(CR4)—Method B

[0301]

[0302] (E,E)-2-Benzylamido-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile (Example 12, 0.028 g, 0.052 mmol) was treated with 60 μlof a 1M THF solution of tetra-n-butylammonium fluoride in 2 ml of dryTHF for 0.5 h at 20° C. After evaporation the compound was dissolved in5 ml of chloroform-methanol, 20:1, passed through silica gel and washedwith chloroform-methanol, 20:1. The residue was purified by HPLCchromatography (MeCN-H₂O, 60:40, UV detection at 340 nm) leading to anorange solid (0.010 g, 62%). The analytical data were identical to thecompound prepared as described in Example 8.

Example 14 (E,E)-2-(3,4 Dihydroxybenzylamido)-3-styrylacrylonitrile(CR19)

[0303]

[0304] The compound was prepared as described in Example 3 by addingcinnamaldehyde (0.018 ml, 0.14 mmol) toN-(cyanoacetyl)3,4-dihydroxybenzylamide (Example 2, 0.03 g, 0.14 mmol).Afer refluxing for 2 h and recrystallization from ethanol, a yellowsolid was obtained (0.027 g, 59%). The product gave the followinganalytical data:

[0305] NMR (CD₃COCD₃, δ, ppm): 2.82 (br.s., 2H, (OH)₂), 4.39 (br.s., 2H,NHCH₂Ph), 6.70 (dd, 1H, J 1.9 and 8.2 Hz, H^(6′)), 6.76 (d, 1H, J 8.2Hz, H^(5′)), 6.87 (d, 1H, J 1.9 Hz, H^(2′)), 7.30 (dd, 1H, J 11.3 and15.7 Hz, PhCCHCCN olefinic), 7.47 and 7.73 (2×m, 6H, aromatic protonsand PhCH olefinic), 7.82 (br.s., 1H, NH), 8.04 (dd, 1H, J<0.5 and 11.3Hz, CHCN olefinic).

[0306] MS, m/e (rel. intensity, %):321 (100) [M+H]⁺, 338 (65) [M+NH₄]⁺.

Example 15 (E,E)-2-(3,4Dihydroxybenzylamido)-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile(CR20)

[0307]

[0308] The compound was prepared as described in Example 3 by adding3,4-bis(t-butyldimethylsilyloxy)cinnamaldehyde (Example 11, 0.015 g,0.038 mmol) to N-(cyanoacetyl)3,4-dihydroxybenzylamide (Example 2,0-0079 g, 0.038 mmol). After refluxing for 2 h and recrystallizationfrom ethanol a yellow solid was obtained (0.014 g, 64%). The productgave the following analytical data:

[0309] NMR (CD₃COCD₃, δ, ppm): 0.22 and 0.24 (2×s, 2×6H, Me₂Si+Me₂Si),1.01 and 1.03 (2×s, 2×9H, t-BuSi+t-BuSi), 2.72 (br.s., 2H, (OH)₂), 4.41(br.s, 2H, NHCH₂Ph), 6.68-7.42 (m, 8H, aromatic and olefinic protons),7.75 (br.s., 1H, NH), 8.00 (dd, 1H, J<0.5 and 12.0 Hz, CHCN olefinic).

[0310] MS, m/e (rel. intensity, %); 555 (5) [M+H−CN]⁺, 572 (8)[M+NH₄−CN]⁺, 581 (46) [M+H]⁺, 598 (100) [M+NH₄]⁺.

Example 16 (E,E)-2-(3,4Dihydroxybenzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile (CR21)

[0311]

[0312](E,E)-2-(3,4-Dihydroxybenzylamido)-3-[3,4-bis(t-butyldimethyl-silyloxystyryl)]acrylonitrile(Example 15, 0.026 g, 0.044 mmol) was treated with 60 μl of a 1M THFsolution of tetra-n-butylammonium fluoride in 1.5 ml of dry THF for 0.5h at 20° C. as described in Example 13. After purification, a yellowsolid was obtained (0.006 g, 43%). The product gave the followinganalytical data:

[0313] NMR (CD₃COCD₃, δ, ppm): 4.38 (s, 1H, NHCH₂Ph), 6.67-7.22 (m, 6H,Ph+Ph′), 7.05 (dd, 1H, J 11.8 and 15.5 Hz, PhC═CH olefinic), 7.34 (d,1H, J 15.5 Hz, PhCH olefinic), 7.70 (br.s, 1H, NH), 8.00 (d, 1H, J 11.8Hz, CH═CCN olefinic).

[0314] MS, m/e (rel. intensity, %), 186 (86) [(HO)₂C₆H₃CH═CHCH═CCN]⁺,202 (28), 242 (100) [M+H−C₆H₃(OH)₂]⁺, 353 (13) [M+H]⁺, 370 (6) [M+NH₄]⁺.

Example 17 (E,E)-2-(Benzylamido)-3-styrylacrylonitrile (CR1)

[0315]

[0316] The compound was prepared as described in Example 3 by addingcinnamaldehyde (0.048 ml, 0.38 mmol) to N-(cyanoacetyl)benzylamide(Example 4, 0.066 g, 0.38 mmol). After refluxing for 1 h andrecrystallization from ethanol a white solid was obtained (0.074 g,68%). The product gave the following analytical data:

[0317] NMR (CD₃COCD₃, δ, ppm): 4.55 (s, 1H, NHCH₂Ph), 7.24-7.51 (m, 11H,Ph+Ph′+PhCCHCCN olefinic), 7.72 (br.d, 1H, J 6.5 Hz, CHCN olefinic),7.98 (br.s., 1H, NH), 8.05 (d, 1H, J 11.7 Hz, PhCH olefinic).

[0318] MS, m/e (rel. intensity, %): 289 (100) [M+H]⁺, 306 (92) [M+NH₄]⁺.

Example 18(E,E)-2-(Benzylamido)-3-(3,5-dimethoxy-4hydroxystyryl)acrylonitrile(CR3)

[0319]

[0320] The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.10 g, 0.48 mmol) toN-(cyanoacetyl)benzylamide (Example 4, 0.084 g, 0.48 mmol). Afterrefluxing for 3 h and recrystallization from ethanol, a yellow solid wasobtained (0.10 g, 57%) The product gave the following analytical data:

[0321] NMR (CD₃COCD₃, δ, ppm): 3.90 (s, 6H, (OMe)₂), 4.55 (m, 2H,NHCH₂Ph), 7.08 (br.s, 2H, H²⁺⁶) 7.17 (dd, 1H, J 11.5 and 15.2 Hz,PhCCHCCN olefinic), 7.22-741 (m, 6H, Ph′+PhCH olefinic), 7.90 (br.s.,1H, NH), 8.01 (dd, 1H, J 0.55 and 11.7 Hz, CHCN olefinic)

[0322] MS, m/e (rel. intensity, %): 275 (14) [M+H−CN−MeOH−MeOH]⁺, 307(9) [M+H−CN−MeOH]⁺, 339 (4) [M+H−CN]⁺, 365 (100) [M+H]⁺, 382 (16)[M+NH₄]⁺.

Example 19 N-(Cyanoacetyl)phenylpropylamide (A₅)

[0323]

[0324] The compound was prepared as described in Example 1 by addingmethyl cyanoacetate (0.98 ml, 11.1 mmol) to phenylpropylamine (1.58 ml,11.1 mmol). The compound was distilled in vacuo directly from thereaction mixture (Kugelrohr apparatus (Aldrich), 0.1 mm Hg, T. oven195-200° C.) to give an off-white solid (2.18 g, 97%). The product gavethe following analytical data:

[0325] NMR (CD₃COCD₃, δ, ppm): 1.88 (q, 2H, J 7.3 Hz, PhCCH₂), 2.66 (t,2H, J 7.3 Hz, PhCH₂), 3.28 (s, 2H, CNCH₂), 3.33 (dt, 2H, J 7.3 and 6.6Hz, PhCCCH₂), 6.02 (br.s., 1H, NH), 7.15-7.30 (m, 5H, Ph).

[0326] MS, m/e (rel. intensity, %): 203 (88) [M+H]⁺, 220 (100) [M+NH₄]⁺.

Example 20 N-(Cyanoacetyl)phenylethylamide (A₄)

[0327]

[0328] The compound was prepared as described in Example 1 by addingmethyl cyanoacetate (1.1 ml, 12.4 mmol) to phenylethylamine (1.55 ml,12.4 mmol). The compound was distilled in vacuo directly from thereaction mixture (Kugelrohr apparatus (Aldrich), 0.1 mm Hg, T. oven190-195° C.) to give an off-white solid (2.14 g, 91%). The product gavethe following analytical data:

[0329] NMR (CD₃COCD₃, δ, ppm): 2.80 (t, 2H, J 7.6 Hz, PhCH₂), 3.46(br.t, 2H, J 7.6 Hz, PhCCH₂), 3.54 (s, 2H, CNCH₂), 7.20-7.31 (m, 5H,Ph), 7.51 (br.s., 1H, NH).

[0330] MS, m/e (rel. intensity, %): 189 (100) [M+H]⁺, 206 (99) [M+NH₄]⁺.

Example 21(E,E)-2-(Phenylethylamido)-3-(3,4-dimethoxystyryl)acrylonitrile (CR5)

[0331]

[0332] The compound was prepared as described in Example 3 by adding3,4-dimethoxycinnamaldehyde (Example 6, 0.1 g, 0.52 mmol) toN-(cyanoacetyl)phenylethylamide (Example 20, 0.1 g, 0.52 mmol). Afterrefluxing for 1 h and recrystallization from ethanol a yellow solid wasobtained (0.12 g, 63%). The product gave the following analytical data:

[0333] NMR (CD₃COCD₃, δ, ppm): 2.91 (t, 2H, J 7.5 Hz, Ph′CH), 3.59(br.t, 2H, J 7.5 Hz, Ph′CCH), 3.88, 3.89 (2×s, 2×3H, OCH₃+OCH₃), 7.04(d, 1H, J 8.6 Hz, H5), 7.16 (dd, 1H, J 11.8 and 15.0 Hz, PhC═CHolefinic), 7.20-7.42 (m, 9H, aromatic+olefinic), 7.97 (d, 1H, J 11.8 Hz,CH═CCN olefinic).

[0334] MS, m/e (rel. intensity, %): 363 (100) [M+H]⁺, 380 (34) [M+NH₄]⁺.

Example 22(E,E)-2-(Phenylethylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR8)

[0335]

[0336] The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.1 g, 0.48 mmol) toN-(cyanoacetyl)phenylethylamide (Example 20, 0.091 g, 0.48 mmol). Theresidue was purified by silica gel chromatography (CHCl₃-hexane, 1:1) togive a yellow solid (0.15 g, 83% yield). The product gave the followinganalytical data:

[0337] NMR (CD₃COCD₃, δ, ppm): 2.95 (t, 2H, J 7.6 Hz, CH₂Ph′), 3.62 (m,2H, CH₂CPh′), 3.94 (s, 6H, (OMe)₂), 7.11 (s, 2H, H²⁺⁶), 7.19 (dd 1H, J11.7 and 15.3 Hz, PhCCHCCN olefinic), 7.23-7.36 (m, 5H, Ph′), 7.41 (d,1H, J 15.3 Hz, PhCH olefinic), 7.45 (br.s., 1H, NH), 7.99 (d, 1H, J 11.7Hz, CHCN olefinic).

[0338] MS, m/e (rel. intensity, %): 379 (100) [M+H]⁺, 396 (7) [M+NH₄]⁺.

Example 23(E,E)-2-(Phenylpropylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR9)

[0339]

[0340] The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4 hydroxycinnamaldehyde (0.10 g, 0.48 mmol) toN-(cyanoacetyl)phenylpropylamide (Example 19, 0.097 g, 0.48 mmol). Afterrefluxing for 3 h the residue was purified by silica gel chromatography(CHCl₃-hexane, 1:1) to give a brown solid (0.17 g, 90% yield). Theproduct gave the following analytical data:

[0341] NMR (CD₃COCD₃, δ, ppm): 2.09 (q, 2H, J 7.5 Hz, NHCCH₂CPh′), 2.85(t, 2H, J 7.5 Hz, CH₂Ph′), 3.57 (m, 2H, CH₂CPh′), 4.06 (s, 6H, (OMe)₂),7.24 (s, 2H, H²⁺⁶), 7.32 (dd, 1H, J 11.7 and 15.3 Hz, PhCCHCCNolefinic), 7,33-7.46 (m, 5H, Ph′), 7.53 (d, 1H, J 15.3 Hz, PhCHolefinic), 7.58 (br.s., 1H, NH), 8.11 (d, 1H, J 11.7 Hz, CHCN olefinic).

[0342] MS, m/e (rel. intensity, %): 331 (40), 348 (30), 359 (34), 376(32), 393 (100) [M+H]⁺, 410 (24) [M+NH₄]⁺.

Example 24(E,E)-2-Thioacetamido-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR12)

[0343]

[0344] The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.15 g, 0.72 mmol) to2-cyanothioacetamide (0.073 g, 0.72 mmol). After refluxing for 1 h theresidue was purified on a TLC-plate in hexane-ethyl acetate, 1:1 to givea red solid (0.10 g, 52%). The product gave the following analyticaldata:

[0345] NMR (CD₃COCD₃, δ, ppm): 2.85 (b.rs, OH+NH₂), 3.91 (s, 6H,(OMe)₂), 7.11 (s, 2H, H²⁺⁶), 7.20 (dd, 1H, J 11.6 and 15.1 Hz, PhCCHCCNolefinic), 7.46 (d, 1H, J 15.1 Hz, PhCH olefinic), 8.22 (dd, 1H, J 0.73and 11.6 Hz, CHCN olefinic).

[0346] MS, m/e (rel. intensity, %): 289 (100), 291 (60) [M+H]⁺, 312 (8)[M+Na]⁺.

Example 25(E,E)-2-Acetamido-3-(3,6-dimethoxy-4-hydroxystyryl)acrylonitrile (CR13)

[0347]

[0348] The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.1 g, 0.48 mmol) to2-cyanoacetamide (0.04 g, 0.48 mmol). After refluxing for 3 h andrecrystallization from ethanol an orange solid was obtained (0.083 g,63%). The product gave the following analytical data:

[0349] NMR (CD₃COCD₃, δ, ppm): 2.82-2.88 (br.s., OH+NH₂), 3.90 (s, 6H,(OMe)₂), 7.08 (s, 2H, H²⁺⁶), 7.16 (dd, 1H, J 11.6 and 15.1 Hz, PhCCHCCNolefinic), 7.38 (d, 1H, J 15.1 Hz, PhCH olefinic), 7.96 (dd, 1H, J 0.73and 11.6 Hz, CHCN olefinic).

[0350] MS, m/e (rel. intensity, %): 275 (100) [M+H]⁺, 292 (28) [M+NH₄]⁺.

Example 26(E,E)-2-Carboxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile (CR14)

[0351]

[0352] The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.15 g, 0.72 mmol) to cyanoaceticacid (0.061 g, 0.72 mmol). After refluxing for 1 h and recrystallizationfrom ethanol a yellow solid was obtained (0.15 g, 75%). The product gavethe following analytical data:

[0353] NMR (CD₃COCD₃, δ, ppm); 3.00 (br.s, OH), 3.91 (s, 6H, (OMe)₂),7.12 (s, 2H, H²⁺⁶), 7.21 (dd, 1H, J 11.6 and 15.1 Hz, PhCCHCCNolefinic), 7.50 (d, 1H, J 15.1 Hz, PhCH olefinic), 8.04 (dd, 1H, J 0.73and 11.6 Hz, CHCN olefinic).

[0354] MS, m/e (rel. intensity, %): 276 (66) [M+H]⁺, 293 (100) [M+NH₄]⁺.

Example 27(E,E)-2-Carbomethoxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitnile(CR15)

[0355]

[0356] The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.15 g, 0.72 mmol) to methylcyanoacetate (0.064 ml, 0.72 mmol). After refluxing for 1 h andrecrystallization from ethanol an orange solid was obtained (0.2 g,90%). The product gave the following analytical data:

[0357] NMR (CD₃COCD₃, δ, ppm): 2.84 (br.s., OH), 3.84 (s, 3H, COOMe),3.91 (s, 6H, (OMe)₂), 7.12 (s, 2H, H²⁺⁶), 7.21 (dd, 1H, J 11.6 and 15.1Hz, PhCCHCCN olefinic), 7.53 (d, 1H, J 15.1 Hz, PhCH olefinic), 8.05(dd, 1H, J 0.73 and 11.6 Hz, CHCN olefinic).

[0358] MS, m/e (rel. intensity, %): 290 (100) [M+H]⁺, 307 (99) [M+NH₄]⁺.

Example 28(E,E)-2-Acetamido-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile(CR16)

[0359]

[0360] The compound was prepared as described in Example 3 by adding3,4-bis(t-butyldimethylsilyloxy)cinnamaldehyde (Example 11, 0.15 g, 0.38mmol) to 2-cyanoacetamide (0.032 g, 0.38 mmol). After refluxing for 0.5h, purification by silica gel chromatography (hexane-EtOAc, 5:1)provided a crystallizing oil (0.10 g, 57%).

[0361] NMR (CD₃COCD₃, δ, ppm): 0.22 and 0.24 (2×s, 2×6H, Me₂Si+Me₂Si),1.01 and 1.03 (2×s, 2×9H, t-BuSi+t-BuSi), 7,01, 7.23-7.29 (m, 3H,aromatic), 7.11 (dd, 1H, J 11.9 and 15.3 Hz, PhC═CH olefinic), 7.40 (d,1H, J 15.3 Hz, PhCH olefinic), 7.98 (d, 1H, J 11.9 Hz, CH═CCN olefinic).

[0362] MS, m/e (rel. intensity, %): 459 (100) [M+H]⁺, 476 (89) [M+NH₄]⁺.

Example 29 (E,E)-2-Acetamido-3-(3,4-dihydroxystyryl)acrylonitrile (CR17)

[0363]

[0364](E,E)-2-Acetamido-3-(3,4-bis(t-butyldimethylsilyloxystyryl))acrylonitrile(Example 28, 0.1 g, 0.22 mmol) was treated with an excess of a 1M THFsolution of tetra-n-butylammonium fluoride in benzene for 0.5 h at 20°C. as described in Example 13. After purification, a yellow solid wasobtained (0.04 g, 85%). The product gave the following analytical data:

[0365] MS, m/e (rel. intensity, %): 231 (83) [M+H]⁺, 248 (100) [M+NH₄]⁺.

Example 30 N-(Cyancacetyl)β-ethanalamide (A₁₁)

[0366]

[0367] To β-ethanolamine (1.37 ml, 22.6 mmol), methyl cyanoacetate wasadded (2.0 ml, 22.6 mmol). The reaction was heated for 30 h at 100° C.Cooling gave a brown solid which was recrystallized from ethanol to give2.10 g of the product (71%). The product gave the following analyticaldata:

[0368] MS, m/e (rel. intensity, %):129 (30) [M+H]⁺, 146 (100) [M+NH₄]⁺.

Example 31(E,E)-2-(β-Ethanolamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR24)

[0369]

[0370] To 3,5-dimethoxy-4-hydroxycinnamaldehyde (0.018 g, 0.086 mmol),N-(cyanoacetyl)β-ethanolamide (Example 30, 0.010 g, 0.086 mmol) wasadded. After refluxing for 2 h and purification on silica gel,CHCl₃-MeOH, 5:1, a yellow solid was obtained. (0.024 g, 87%). Theproduct gave the following analytical data:

[0371] NMR (CD₃COCD₃, δ, ppm): 3.47, 3.67 (2×m, 4H, NHCH₂+CH₂OH), 3.90(s, 6H, OCH₃+OCH₃), 7.07 (br.s, 2H, H²⁺⁶), 7.16 (dd, 1H, J 11.7 and 15.2Hz, PhC═CH olefinic), 7.31 (br.s, 1H, NH), 7.38 (d, 1H, J 15.2 Hz, PhCHolefinic), 7.97 (d, 1H, J 11.7 Hz, CH═CCN olefinic).

[0372] MS, m/e (rel. intensity, %): 319 (70) [M+H]⁺, 341 (100) [M+Na]⁺.

Example 32 (E,E)-2-(Benzylamido)-3-(4-nitrostyryl)acrylonitrile (CR27)

[0373]

[0374] The compound was prepared as described in Example 3 by adding4-nitrocinnamaldehyde (0.022 g, 0.12 mmol) to N-(cyanoacetyl)benzylamide(Example 4, 0.022 g, 0.12 mmol). After refluxing for 1 h, the productwas purified by silica gel chromatography (CHCl₃-MeOH, 5:1) to give ayellow solid (0.033 g, 81%) The product gave the following analyticaldata:

[0375] NMR (CD₃COCD₃, δ, ppm): 4.56 (br.s, 2H, NHCH₂), 7.24-7.38 (m, 6H,Ph′+NH), 7.47 (dd, 1H, J 11.1 and 15.2 Hz, PhC═CH olefinic), 7.62 (d,1H, J 15.2 Hz, PhCH olefinic), 8.02, 8.32 (2×br.d, 4H, J 8.8 and 8.3 Hz,Ph), 8.08 (d, 1H, J 11.1 Hz, CH═CCN olefinic).

[0376] MS, m/e (rel. intensity, %): 334 (100) [M+H]⁺, 351 (16) [M+NH₄]⁺,356 (28) [M+Na]⁺.

Example 33(E,E)-2-(3,4-Dihydroxybenzylamido)-3-(4-nitrostyryl)acrylonitrile(CR28)

[0377]

[0378] The compound was prepared as described in Example 3 by adding4-nitrocinnamaldehyde (0.009 g, 0.05 mmol) toN-(cyanoacetyl)3,4-dihydroxybenzylamide (Example 2, 0.010 g, 0.05 mmol).After refluxing for 2 h and recrystallization from ethanol a yellowsolid was obtained (0.007 g, 39%). The product gave the followinganalytical data:

[0379] NMR (CD₃COCD₃, δ, ppm): 2.81, 2.83 (2×br.s, 2H, OH+OH), 4.39(br.s, 2H, NHCH₂), 6.69 (br.d, 1H, J<0.5 and 7.6 Hz, H^(6′)), 6.76 (d,1H, J 7.6 Hz, H^(5′)), 6.86 (br.d, 1H, J<0.5 Hz, H^(2′)), 7.47 (dd, 1H,J 11.7 and 15.2 Hz, PhC═CH olefinic), 7.61 (d, 1H, J 15.2 Hz, PhColefinic), 7.91 (br.s, 1H, NH), 8.02, 8.31 (2×br.d, 4H, J 8.2 and 8.8Hz, Ph), 8.06 (d, 1H, J 11.7 Hz, CH═CCN olefinic).

[0380] MS, m/e (rel. intensity, %): 331 (21) [M−OH−OH]⁺, 348 (47)[M−OH]⁺, 366 (100) [M+H]⁺, 383 (97) [M+NH₄]⁺.

Example 34(E,E)-2-(1-Amino-2,2-dicyanoethenyl)-3-(4-nitrostyryl)acrylonitrile(CR29)

[0381]

[0382] The compound was prepared as described in Example 3 by adding4-nitrocinnamaldehyde (0.051 g, 0.29 mmol) to2-amino-1-propene-1,1,3-tricarbonitrile (0.038 g, 0.29 mmol). Afterrefluxing for 4 h and recrystallization from ethanol a yellow solid wasobtained (0.08 g, 51%). The product gave the following analytical data:

[0383] NMR (CD₃COCD₃, δ, ppm): 7.54 (dd, 1H, J 11.1 and 15.8 Hz, PhC═CHolefinic), 7.67 (d, 1H, J 15.8 Hz, PhCH olefinic), 7.99 (d, 1H, J 11.1Hz, CH═CCN olefinic), 8.08, 8.32 (2×br.d, 4H, J 8.8 and 8.8 Hz, Ph).

[0384] MS, m/e (rel. intensity, %): 309 (100) [M+NH₄]⁺, 314 (67)[M+Na]⁺.

Example 35 Effect of CR4 Upon Normal Bone Marrow Differentiation inCulture

[0385] The CFU-GEMM assay was performed according to Fauser and Messner(1978, Blood, 52(6) 143-8) and Messner and Fausser (1980, Blut, 41(5)327-33) with some variations. In brief, heparinized bone marrow cellswere layered over Percoll (1.077 gm/ml) (Pharmacia Fine Chemical,Piscataway N.J.) and centrifuged at 400 g at 4° C. for 10 minutes toremove neutrophils and RBCs. The fractionated BM cells at 2×10⁵ cells/mlwere cultured in IMDM (OCI, Toronto) containing 0.9% (vol/vol)methylcellulose supplementd with 30% FCS (Cansera Rexdale, ON.) ornormal human plasma, a cocktail of cytokines containing G-CSF (10 ng/ml,Amgen), IL-3 (40 U/ml, Immunex), MGF (50 ng/ml, Immunex), Erythropoietin(2 u/ml, Epprex) or TPO (10 ng/ml, Amgen), 5×10⁻⁵M β-2-mercaptoethanoland the specified concentration of CR4. The culture mixture was platedin 1 ml volumes into 35 mm petri dishes and incubated at 37° C., 5% CO₂in a humidified atmosphere. All cultures were evaluated at 14 days forthe number of BFU-E colonies (defined as aggregates of more than 500hemoglobinized cells or, 3 or more erythroid subcolonies), OFU-GMcolonies (defined as granulocyte or monocyte-macrophage cells or both),CFU-Meg colonies (comprising 4 or more megakaryocytes) and CFU-GEMMcolonies (a mixed population comprising of all elements).

[0386] The results shown in FIG. 1 demonstrate that CR4 displayednegligible toxicity upon normal bone marrow at doses up to 5 μM. At 10μM CR4 began to cause some inhibition of BFU-E colony formation, but atthe same time significantly stimulated CFU-GM colony numbers.

Example 36 Killing of Philadelphia Positive Acute Lymphablastic Leukemiaby Low-dose CR4 in Culture

[0387] Ph+ ALL cells were plated in 1 ml volumes, in the absence ofexogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing alpha MEM (Gibco) plus 10% FCS (Cansera Rexdale, ON.) in 0.9%(vol/vol) methylcellulose (Fluka, Switzerland) with the indicatedconcentrations of CR4. Cultures were set at 37° C., 5% CO₂ in ahumidified atmosphere. Colonies consisting of more than 20 cells werecounted at 12 days or earlier using an inverted microscope.

[0388] The results shown in FIG. 2 demonstrate that CR4 effected asignificant inhibition of Ph+ ALL cell proliferation and survival at lownanomolar doses (35-100). CR4 has no effect upon normal cells atequivalent concentrations.

Example 37 Killing of Philadelphia Positive Z119 Acute LymphoblasticLeukemia Cells by Low-dose CR4 in Culture

[0389] Z119 cells were plated in 1 ml volumes at a density of 1×10⁴celIs/ml, in the absence of exogenous growth factors, into 35 mm petridishes (Nunc, Gibco) containing IMDM (OCI, Toronto) plus 20% FCS(Cansera Rexdale, ON.) in 0.9% (vol/vol) methylcellulose (Fluka,Switzerland) with the indicated concentration of CR4. Cultures were setat 37° C., 5% CO₂ in a humidified atmosphere. Colonies consisting ofmore than 20 cells were counted at 7 days or earlier using an invertedmicroscope.

[0390] The results shown in FIG. 3 demonstrate that CR4 effected asignificant inhibition of Z119 ALL cell proliferation and survival atlow nanomolar doses. CR4 has no effect upon normal cells at equivalentconcentrations.

Example 38 Killing of AML-3 Acute Myeloid Leukemia Cells by Low-dose CR4in Culture

[0391] OCI-AML-3 cells were plated in 35 mm petri dishes (Nunc, Gibco)in 1 ml volumes at a density of 3.3×10³ cells/ml, in the absence ofexogenous growth factors, containing alpha MEM plus 20% FCS (Cansera,Rexdale Ont.), and 0.9% (vol/vol) methylcellulose (Fluka, Switzerland)and the indicated concentrations of CR4. Cell cultures were incubated ina humidified atmosphere at 37° C. with 5% CO₂. Colonies containing morethan 20 cells were scored, using an inverted microscope, at 5-6 days.

[0392] The results shown in FIG. 4 demonstrate that CR4 effected acomplete inhibition of AML-3 cell proliferation and survival atnanomolar concentrations (300-600 nM). CR4 has no effect upon normalcell survival at equivalent concentrations.

Example 39 Killing of Ly-MN Lymphoma Cells by Low-dose CR4 in Culture

[0393] Ly-MN cells were plated in 1 ml volumes, in the absence ofexogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing IMDM (OCI, Toronto) plus 20% human cord blood plasma in 0.9%(vol/vol) methylcellulose (Fluka, Switzerland) and the indicatedconcentrations of CR4. Cultures were set at 37° C., 5% CO₂ in ahumidified atmosphere. Colonies consisting of more than 20 cells werecounted at 5 days or earlier using an inverted microscope.

[0394] The results shown in FIG. 5 demonstrate that CR4 significantlyinhibited cell proliferation and survival at nanomolar doses, andeffected a inhibition by 2.5 μM. CR4 has no effect upon normal cells atequivalent concentrations.

Example 40 Killing of Primary Juvenile Myelo-Monocytic Leukemia Cells byCR4 in Culture

[0395] Heparinized bone marrow cells from a JMML patient were layeredover Percoll (1.077 gm/ml) (Pharmacia Fine Chemical, Piscataway N.J.)and centrifuged at 400 g at 4° C. for 10 minutes to remove neutrophilsand RBCs. Cells were further fractionated and purified on Miltenyi MScolumns (Miltenyi Biotec GmbH, Germany) to acquire an early progenitorpopulation of CD34⁺ cells. The fractionated BM CD34⁺ cells at a densityof 1×10⁴ cells/ml were cultured in IMDM (OCI, Toronto) containing 0.9%(vol/vol) methylcellulose supplemented with 30% FCS (Cansera Rexdale,ON.) with the indicated concentration of CR4. The culture mixture wasplated in 1 ml volumes into 35 mm petri dishes and incubated at 37° C.,5% CO₂ in a humidified atmosphere. Colonies consisting of more than 20cells were counted at 12 days or earlier using an inverted microscope.

[0396] The results shown in FIG. 6 demonstrate that CR4 displayedmoderate killing ability with primary JMML cells, with 80-90 percentinhibition achieved by 5 μM concentrations.

Example 41 Killing of OCI-LY2 Lymphoma Cells by Low-dose CR4 in Culture

[0397] OCI-LY2 cells were plated in 1 ml volumes, in the absence ofexogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing IMDM (OCI, Toronto) plus 20% human cord blood plasma in 0.9%(vol/vol) methylcellulose (Fluka, Switzerland) and the indicated dosesof CR4. Cultures were set at 37° C., 5% CO₂ in a humidified atmosphere.Colonies consisting of more than 20 cells were counted at 5 days orearlier using an inverted microscope.

[0398] The results shown in FIG. 7 demonstrate that CR4 significantlyinhibited cell proliferation and survival at high nanomolar to lowmicromotar doses (90% at 2.5 μM). CR4 has no effect upon normal cells atequivalent concentrations.

Example 42 Killing of Philadelphia Positive ALL Cells by CR17 and CR21in Culture

[0399] ALL cells were plated in 1 ml volumes, in the absence ofexogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing alpha MEM (Gibco) plus 20% FCS (Cansera Rexdale, ON.) in 0.9%(vol/vol) methylcellulose (Fluka, Switzerland) and the indicatedconcentrations of compound Cultures were set at 37° C., 5% CO₂ in ahumidified atmosphere Colonies consisting of more than 20 cells werecounted at 12 days or earlier using an inverted microscope.

[0400] The results shown in FIG. 8 demonstrate that CR17 displayedsignificant inhibition of cell growth at 1-2.5 μM concentrations. CR21inhibited cell growth at 5 μM.

Example 43 Killing of Philadelphia Positive ALL Cells by CR17 and CR21in Culture

[0401] ALL cells were plated in 1 ml volumes, in the absence ofexogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing alpha MEM (Gibco) plus 20% FCS (Cansera Rexdale, ON) in 0.90%(vol/vol) methylcellulose (Fluka, Switzerland) and the indicatedconcentrations of compound. Cultures were set at 37° C., 5% CO₂ in ahumidified atmosphere. Colonies consisting of more than 20 cells werecounted at 12 days or earlier using an inverted microscope.

[0402] The results shown in FIG. 9 demonstrate that CR17 and CR21 bothdisplayed significant inhibition of cell growth at 1-2.5 μMconcentrations.

Example 44 Killing of Philadelphia Positive ALL Cells by CR24 in Culture

[0403] ALL cells were plated in 1 ml volumes, in the absence ofexogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing alpha MEM (Gibco) plus 20% FCS (Cansera Rexdale, ON.) in 0.9%(vol/vol) methylcellulose (Fluka, Switzerland) and the indicatedconcentrations of CR24. Cultures were set at 37° C., 5% CO₂ in ahumidified atmosphere. Colonies consisting of more than 20 cells werecounted at 9 days or earlier using an inverted microscope.

[0404] The results shown in FIG. 10 demonstrate that CR24 was effectiveagainst Ph+ ALL cells at concentrations as low as 0.5 μM, demonstratinga virtually complete inhibition of cell growth between 2.5 and 5 μM.

Example 45 Killing of Philadelphia Positive ALL Cells by CR19 in Culture

[0405] ALL cells were plated in 1 ml volumes, in the absence ofexogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing alpha MEM (Gibco) plus 20% FCS (Cansera Rexdale, ON.) in 0.9%(vol/vol) methylcellulose (Fluka, Switzerland) and the indicatedconcentrations of CR19. Cultures were set at 37° C., 5% CO₂ in ahumidified atmosphere. Colonies consisting of more than 20 cells werecounted at 9 days or earlier using an inverted microscope.

[0406] The results shown in FIG. 11 demonstrate that CR19 was highlyeffective against Ph+ ALL cells at nanomolar concentrations between 250and 500 mM.

Example 46 Effect of CR19 On Normal Bone Marrow Differentiation inCulture

[0407] The CFU-GEMM assay was performed according to Fauser and Messner(1978, Blood, 52(6) 1243-8) and Messner and Fausser (1980, Blut, 41(5)327-33) with some variations. In brief, heparinized bone marrow cellswere layered over Percoll (1.077 gm/ml) (Pharmacia Fine Chemical,Piscataway N.J.) and centrifuged at 400 g at 4° C. for 10 minutes toremove neutrophils and RBCs. The fractionated BM cells at 2×10⁵ cells/mlwere cultured in IMDM (OCI, Toronto) containing 0.9% (vol/vol)methylcellulose supplementd with 30% FCS (Cansera Rexdale, ON.) ornormal human plasma, a cocktail of cytokines containing G-CSF (10 ng/ml,Amgen), IL-3 (40 U/ml, Immunex), MGF (50 ng/ml, Immunex), Erythropoietin(2 u/ml, Epprex) or TPO (10 ng/ml, Amgen), 5×10⁻⁵M β-2-mercaptoethanoland the specified concentrations of CR19. The culture mixture was platedin 1 ml volumes into 35 mm petri dishes and incubated at 37° C., 5% CO₂in a humidified atmosphere. All cultures were evaluated at 14 days forthe number of BFU-E colonies (defined as aggregates of more than 500hemaglobinized cells or, 3 or more erythroid subcolonies) and CFU-Ccolonies (defined as granulocyte or monocyte-macrophage cells or both).

[0408] The results shown in FIG. 12 demonstrate that CR19 displayedsignificant inhibition of the development of BFU-E colonies at 2.5 μM,although at this concentration it also boosted CFU-C colony formation.At 5 μM the stimulatory effect disappeared and BFU-E colonies werevirtually absent.

Example 47 Effect of CR24, CR17 and CR21 On Normal Bone MarrowDifferentiation

[0409] The CFU-GEMM assay was performed according to Fauser and Messner(1978, Blood, 52(6) 1243-8) and Messner and Fausser (1980, Blut, 41(5)327-33) with some variations (British Journal of Haematology, 1992, 80,p40-48). In brief, heparinized bone marrow cells were layered overPercoll (1.077 gm/ml) (Pharmacia Fine Chemical, Piscataway N.J.) andcentrifuged at 400 g at 4° C. for 10 minutes to remove neutrophils andRBCs. The fractionated BM cells at 2×10⁵ cells/ml were cultured in IMDM(OCI, Toronto) containing 0.9% (vol/vol) methylcellulose supplementdwith 30% FCS (Cansera Rexdale, ON.) or normal human plasma, a cocktailof cytokines containing G-CSF (10 ng/ml, Amgen), IL-3 (40 U/ml,Immunex), MGF (50 ng/ml, Immunex), Erythropoietin (2 u/ml, Epprex) orTPO (10 ng/ml, Amgen), 5×10⁻⁵M β-2-mercaptoethanol and the specifiedconcentration of test compound. The culture mixture was plated in 1 mlvolumes into 35 mm petri dishes and incubated at 37° C., 5% CO₂, in ahumidified atmosphere. All cultures were evaluated at 14 days for thenumber of BFU-E colonies (defined as aggregates of more than 500hemoglobinized cells or, 3 or more erythroid subcolonies) and CFU-Ccolonies (defined as granulocyte or monocyte-macrophage cells or both),

[0410] The results shown in FIG. 13 demonstrate that CR24 displayedminimal inhibition of bone marrow colony formation at either 10 or 20 μMconcentrations, whereas both CR17 and CR21 caused inhibition of BFU-Ecolony formation at the higher 20 μM dose.

Example 48 In vitro Purging of Normal Bone Marrow with CR4

[0411] Heparinized bone marrow cells were layered over Percoll (1.077gm/ml) (Pharnacia Fine Chemical, Piscataway N.J.) and centrifuged at 400g at 4° C. for 10 minutes to remove neutrophils and RBCs.

[0412] For the purging process, the cells were resuspended at 1×10⁶/mlin complete medium with or without 50 μM CR4. The cells were incubatedwith the CR4 for two and a half hours at 37° C., 5% CO₂. At the end ofthis period the cells were thoroughly washed in medium to remove CR4 andthen cultured at 2×10⁵ cells/ml in IMDM (OCI, Toronto) containing 0.9%(vol/vol) methylcellulose supplemented with 30% FCS (Cansera Rexdale,ON.) or normal human plasma, a cocktail of cytokines containing G-CSF(10 ng/ml, Amgen), IL-3 (40 U/ml, Immunex), MGF (50 ng/ml, Immunex),Etryhropoietin (2 u/ml, Epprex) or TPO (10 ng/ml, Amgen) and 5×10⁻⁵Mb-2-mercaptoethanol. The culture mixture was plated in 1 ml volumes into35 mm petri dishes and incubated at 37° C., 5% CO₂ in a humidifiedatmosphere. All cultures were evaluated at 14 days for the number ofBFU-E colonies (defined as aggregates of more than 500 hemaglobinizedcells or, 3 or more erythroid subcolonies), CFU-C colonies (defined asgranulocyte or monocyte-macrophage cells or both) and CFU-GEMM colonies(a mixed population comprising of all elements).

[0413] The results shown in FIG. 14 demonstrate that two and a halfhours exposure to 50 μM CR4 did not result in significant inhibition ofcolony formation. While a slight drop in BFU-E colonies occurred, CFU-Ccolony numbers actually increased significantly.

Example 49 In Vitro Purging of Z119 Acute Lymphoblastic Leukemia withCR4

[0414] For the purging assay, the cells were resuspended in completemedium with or without CR4 as indicated and incubated at 37° C., 5%CO₂for 0-5 hours. Cells were then washed thoroughly with medium to removethe CR4, resuspended and plated in 1 ml volumes, in the absence ofexogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing alpha MEM (Gibco) plus 20% FCS (Cansera Rexdale, ON.) in 0.9%(vol/vol) methylcellulose (Fluka, Switzerland). Cultures were set at 37°C., 5% CO₂ in a humidified atmosphere. Colonies consisting of more than20 cells were counted at 9 days or earlier using an inverted microscope.

[0415] The results shown in FIG. 15 demonstrate that CR4 demonstratedrapid killing of Z119 cells at the concentrations examined. 50 μM CR4displayed a complete inhibition after only 2.5 hours exposure, while 85%killing could be achieved with 25 μM CR4 over the same time period. Alonger five hour exposure of the cells to 25 μM CR4 resulted in acomplete ablation of subsequent cell growth.

Example 50 In vitro Purging of OCI-Ly2 Lymphoma Cells with CR4

[0416] For the purging assay, the cells were resuspended in completemedium with or without CR4 as indicated and incubated at 37° C., 5%CO₂for 0-5 hours. Cells were then washed thoroughly with medium to removeCR4, resuspended and plated in 1 ml volumes at 5×10³ cellls/ml, in theabsence of exogenous growth factors, into 35 mm petri dishes (Nunc,Gibco) containing IMDM (OCI, Toronto) plus 20% human cord blood plasmain 0.9% (vol/vol) methylcellulose (Fluka, Switzerland). Cultures wereset at 37° C., 5% CO₂ in a humidified atmosphere. Colonies consisting ofmore than 20 cells were counted at 5 days or earlier using an invertedmicroscope.

[0417] The results shown in FIG. 16 demonstrate that CR4 demonstratedsignificant killing (90%) of OCI-Ly2 cells at 25-50 μM after only 5hours exposure. The lower 12.5 μM dose tested also achieved significantkilling in the same time period.

Example 51 In vitro Purging of OCI-AML-3 Acute Meyloid Leukemia Cellswith CR4

[0418] For the purging assay, the cells were resuspended in completemedium with or without CR4 as indicated and incubated at 37° C., 5%CO₂for 0-5 hours. Cells were then washed thoroughly with medium to removeCR4, resuspended and plated in 1 ml volumes at 5×10³ cells/ml, in theabsence of exogenous growth factors, into 35 mm petri dishes (Nunc,Gibco) containing alpha MEM (Gibco) plus 10% FCS (Cansera Rexdale, ON.)in 0.9% (vol/vol) methylcellulose (Fluka, Switzerland). Cultures wereset at 37° C., 5% CO₂ in a humidified atmosphere. Colonies consisting ofmore than 20 cells were counted at 5 days or earlier using an invertedmicroscope.

[0419] The results shown in FIG. 17 demonstrate that CR4 demonstratedsignificant killing of OCI-AML-3 cells at 50 μM after only 2.5-5 hoursexposure. The lower 25 μM dose tested also achieved significant killingin the same time period.

Example 52 In vitro Purging of Ramos B Cell Burkitt's Lymphoma Cellswith CR4

[0420] For the purging assay, the cells were resuspended in completemedium with or without CR4 as indicated and incubated at 37° C., 5%CO₂for 0-5 hours. Cells were then washed thoroughly with medium to removethe CR4, resuspended and plated in 1 ml volumes, in the absence ofexogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing RPMI 1640 (Gibco) plus 10% FCS (Cansera Rexdale, ON.) in 0.9%(vol/vol) methylcellulose (Fluka, Switzerland). Cultures were set at 37°C., 5% CO₂ in a humidified atmosphere. Colonies consisting of more than20 cells were counted at 12 days or earlier using an invertedmicroscope.

[0421] The results shown in FIG. 18 demonstrate that CR4 demonstratedsignificant killing (70%) of Ramos cells at 50 μM after 5 hoursexposure.

Example 53 Killing of HuNS1 Multiple Myeloma by CR4

[0422] HuNS1 cells were plated in 35 mm petri dishes (Nunc, Gibco) in 1ml volumes at a density of 1×10⁴ cells/ml, in the absence of exogenousgrowth factors, containing alpha MEM plus 20% FCS (Cansera, RexdaleOnt.), and 0.9% (vol/vol) methylcellulose (Fluka, Switzerland) and theindicated concentrations of CR4. Cell cultures were incubated in ahumidified atmosphere at 37° C. with 5% CO₂. Colonies containing morethan 20 cells were scored, using an inverted microscope, at 56 days.

[0423] The results shown in FIG. 19 demonstrate that CR4 significantlyinhibited cell proliferation and survival at high nanomolar to lowmicromolar doses (>90% at 2.5 μM). CR4 has no effect upon normal cellsat equivalent concentrations.

Example 54 In vivo Treatment of Philadelphia Positive AcuteLymphoblastic Leukemia in NOD-SCID mice

[0424] NOD-SCID mice were irradiated (350 rads) and injected with 5×10⁶Philadelphia positive Z119 Acute lymphoblastic leukemia cells. After 24hours Alzet micro-osmotic pumps (Alza Corp. Paolo Alto, Calif.) wereimplanted subcuntaneously, containing either 20 mM solution of CR4 in50% DMSO/medium or 50% DMSO/medium alone. Alzet 2001 pumps wereutilized, holding a total volume of 200 μl and releasing 1 μl per hourover 7-10 days. Pumps were replaced after 7 days. Each mouse received adaily dose of 0.154 mg of CR4.

[0425] After 14 (FIG. 20A) and 21 (FIG. 20B) days mice were sacrificedand bone marrow extracted from the fore and hind limbs. Single cellsuspensions were prepared, red blood cells lysed and the samples stainedwith PE-labelled isotype, anti-human CD₁₉ and anti-human HLA-DRantibodies to detect the presence of Z119 cells. These antibodies do notcross react with murine cells.

[0426] At d14, bone marrow cell cultures were also performed to assessthe presence of Z119 cells 5×10⁴ BM cells were cultured in IMDM (OCI,Toronto) containing 0.9% (vol/vol) methylcellulose supplemented with 30%serum consisting of a 1:1 mixture of FCS (Cansera Rexdale, ON.) andnormal human plasma. No cytokines are added. Under these conditionsthere is no growth of murine cells. The culture mixture was plated in 1ml volumes into 35 mm petri dishes and incubated at 37° C., 5% CO₂ in ahumidified atmosphere. All cultures were evaluated at 9 days for thenumber of ALL colonies.

[0427] The results shown in FIGS. 20A and 20B demonstrate that at bothday 14 and 21 sacrifices, a significant reduction in ALL infiltration ofthe bone marrow was observed in all the mice treated with CR4 relativeto the OMSO treated control mice.

[0428] In control animals, massive infiltration of the spleen, liver andkidney was observed, as well as the presence of ALL cells in theperipheral blood. In addition to a 90% reduction in the infiltration ofALL cells into the bone marrow, treatment with CR4 reduced ALLinfltration of the organs and blood to below detectable levels.

[0429] Thus, CR4 was highly effective against a variety of cancer cells,including acute lymphoblastic leukemia, Philadelphia positive ALL, acutemyeloid leukemia, myeloma and B-lineage lymphoma, at concentrationsranging from 50 nM to 5 μM. At the same time, minimal toxicity was seenwhen normal cells were incubated in the presence of CR4 untilconcentrations of 10-20 μM or greater were achieved. CR4 wasparticularly active against bcr-abl transformed Philadelphia positivecells, achieving >90% wipeout at concentrations as low as 40 nM. CR4 wasalso highly effective in high dose (25-50 μM) in vitro purging assaysagainst Philadelphia positive ALL, AML and lymphoma, causing >90%inhibition of growth with a 2.5 to 5 hour exposure time. Over identicaldoses and times normal bone marrow growth and differentiation wereunaffected. CR4 showed a combination of high level toxicity to cancercells with minimal non-specific cytotoxic damage.

[0430] CR4 was also highly effective in a whole animal model (Example54). The compound demonstrated good retention characteristics, stillbeing detectable in the blood 30 minutes after I.V. injection. In amurine model of human Ph+ ALL, CR4 caused a greater than 90 percentreduction in ALL infiltration of bone marrow within a two week period,reducing the presence of infiltrating ALL cells in liver, kidney, spleenand peripheral blood below detection level. In contrast, control micetreated with vehicle alone demonstrated massive infiltration of allthese organs. No evidence of non-specific toxicity was observed.

Example 55 In vitro Purging of Normal Bone Marrow with CR11

[0431] Heparinized bone marrow cells were layered over Percoll (1.077gm/ml) (Pharmacia Fine Chemical, Piscataway N.J.) and centrifuged at 400g at 4° C. for 10 minutes to remove neutrophils and RBCs.

[0432] For the purging process, the cells were resuspended at 1×10⁶/mlin complete medium with or without 50 μM CR11. The cells were incubatedwith the tryrphostin for seven hours at 37° C., 5% CO₂. At the end ofthis period the cells were thoroughly washed in medium to remove CR11and then cultured at 2×10⁵ cells/ml in IMDM (OCI, Toronto) containing0.9% (vol/vol) methylcellulose supplementd with 30% FCS (CanseraRexdate, ON.) or normal human plasma, a cocktail of cytokines containingG-CSF (10 ng/ml, Amgen), IL-3 (40 U/ml, Immunex), MGF (50 ng/ml,Immunex), Erythropoietin (2 u/ml, Epprex) or TPO (10 ng/ml, Amgen) and5×10⁻⁵M β-2-mercaptoethanol. The culture mixture was plated in 1 mlvolumes into 35 mm petri dishes and incubated at 37° C., 5% CO₂ in ahumidified atmosphere. All cultures were evaluated at 14 days for thenumber of BFU-E colonies (defined as aggregates of more than 500hemoglobinized cells or, 3 or more erythroid subcolonies), CFU-Ccolonies (defined as granulocyte or monocyte-macrophage cells or both)and CFU-GEMM colonies (a mixed population comprising of all elements).

[0433] The results shown in FIG. 21 demonstrate that seven hoursexposure to 50 μM CR11 did not result in any significant inhibition ofcolony formation. BFU-E, CFU-GEMM and CFU-C colonies were all normal.

Example 56 In vitro Purging of Philadelphia Positive Acute LymphoblasticLeukemia with CR11

[0434] For the purging assay, the cells were resuspended in completemedium with or without CR11 as indicated and incubated at 37° C., 5%CO₂for 0-7 hours. Cells were then washed thoroughly with medium to removethe CR11, resuspended and plated in 1 ml volumes, in the absence ofexogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing alpha MEM (Gibco) plus 20% FCS (Cansera Rexdale, ON.) in 0.9%(vol/vol) methylcellulose (Fluka, Switzerland). Cultures were set at 37°C., 5% CO₂ in a humidified atmosphere. Colonies consisting of more than20 cells were counted at 9 days or earlier using an inverted microscope.

[0435] The results shown in FIG. 22 demonstrate that CR11 demonstratedcomplete killing of Ph+ ALL cells at 50 μM after 7 hours exposure.

Example 57 Philadelphia (Ph+) ALL Lines Z119 and Z181 (5×10⁶Cells/point) Were Lysed and Immunoprecipitated with Bcr-Abl Antibody

[0436] The precipitates were washed twice with lysis buffer and oncewith kinase assay buffer, and resuspended in same buffer containingvarying concentrations of CR4. The precipitates were incubated with thedrug for 10 min at room temperature, followed by addition of 10 μCi³³PγATP. The reaction was stopped after 20 min by the addition ofSDS-PAGE reducing sample buffer and separated on an 8-16% SDS-PAGE gel.The products were transferred onto nitrocellulose membrane andvisualized by autoradiography.

[0437] The results shown in FIG. 23 demonstrate that Bcr-Abl kinaseactivity is effectively blocked at concentrations of 1 to 10 μM of theCR4 compound in both Z199 and Z181 ALL cell lines.

Example 58 Philadelphia (Ph+) ALL Line Z119 (5×10⁶ Cells/point) WasPreincubated for 5 Hours with Different Concentrations of CR4 andImmunoprecipitated with Jak2 Antibody

[0438] The cells were lysed in lysis buffer and immunoprecipitated withJak2 antibody. The precipitates were washed twice with lysis buffer andonce with kinase assay buffer, followed by addition of 10 μCi ³³PγATP.The reaction was stopped after 20 min by the addition of SDS-PAGEreducing sample buffer and separated on an 8-16% SDS-PAGE gel. Theproducts were transferred onto nitrocellulose membrane and visualized byautoradiography.

[0439] The results shown in FIG. 24 demonstrate that Jak2 kinaseactivity was dramatically inhibited at a concentration of 6 μM andfurther blocked at higher concentrations.

[0440] While the present invention has been described with reference towhat are presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

[0441] All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

We claim:
 1. A compound of Formula I, and salts, solvates or hydratesthereof:

wherein R¹ and R² are each independently selected from the groupconsisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo; R³ isselected from the group consisting of H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;R⁴ is selected from the group consisting of C(X)R⁵, SO₃Ar, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), P(O)(OH)₂, P(O)(OC₁₋₆alkyl)₂, andC(NH₂)═C(CN)₂; X is selected from O,S, NH and N—C₁₋₆alkyl; R⁵ isselected from the group consisting of NH₂, OH, NH(CH₂)_(p)Ar,NH(CH₂)_(p)OH, (CH₂)_(p)OC₁₋₆alkyl, C₁₋₆alkyl, C₁₋₆alkoxy, NHNH₂,NHC(O)NH₂, NHC(O)C₁₋₆alkoxy, N-morpholino and N-pyrrolidino; and Ar isan aromatic or heteroaromatic group, unsubstituted or substituted with1-4 substituents, independently selected from the group consisting ofOH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo; n is 0 to 4; and p is 1-4. 2.The compound according to claim 1, wherein R¹ and R² are eachindependently selected from the group consisting of H, OH, C₁₋₄alkyl,C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl, SH, S—C₁₋₄alkyl,O—Si(C₁₋₄alkyl)(C₁₋₄alkyl)(C₁₋₄alkyl), NO₂, CF₃, OCF₃ and halo.
 3. Thecompound according to claim 2, wherein R¹ and R² are each independentlyselected from the group consisting H, OH, OCH₃, O—Si(CH₃)₂(^(t)Bu),S—Me, SH and NO₂.
 4. The compound according to claim 3, wherein R¹ andR² are both OH or R¹ and R² are both OCH₃.
 5. The compound according toclaim 4, wherein R¹ is OCH₃ and R² is OH.
 6. The compound according toclaim 1, wherein R³ is selected from the group consisting of H, OH,C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl, N(C₁₋₄alkyl)(C₁₋₄alkyl), SH,S—C₁₋₄alkyl, NO₂ and halo.
 7. The compound according to claim 6, whereinR³ is selected from the group consisting of H, OH, OCH₃, SH, SMe, NO₂and halo.
 8. The compound according to claim 7, wherein R³ is selectedfrom the group consisting of H, OH and OCH₃.
 9. The compound accordingto claim 1, wherein R⁴ is selected from the group consisting of C(X)R⁵and C(NH₂)═C(CN)₂.
 10. The compound according to claim 9, wherein R⁴isC(X)R⁵.
 11. The compound according to claim 10, wherein X is selectedfrom the group consisting of O and S.
 12. The compound according toclaim 10, wherein R⁵ is selected from the group consisting of NH₂, OH,NH(CH₂)_(p)Ar, NH(CH₂)_(p)OH and C₁₋₄alkoxy.
 13. The compound accordingto claim 12, wherein p is 1-3.
 14. The compound according to claim 13,wherein R⁵ is selected from the group consisting of NH₂, OH,NH(CH₂)_(p)Ar, NH(CH₂)_(p)OH and OCH₃.
 15. The compound according toclam 14, wherein p is 1-2.
 16. The compound according to claim 1,wherein Ar is an unsubstituted phenyl group or a phenyl groupsubstituted with 1-4 substituents optionally selected from the groupconsisting of OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo. 17.The compound according to claim 14, wherein Ar is an unsubstitutedphenyl group or a phenyl group substituted with 1-4 substituentsoptionally selected from the group consisting of OH, C₁₋₆alkyl,C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,NO₂, CF₃, OCF₃ and halo.
 18. The compound according to any of claims 16or 17, wherein Ar is an unsubstituted phenyl group or phenyl groupsubstituted with 1-2 substituents optionally selected from the groupconsisting of OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl,N(C₁₋₄alkyl)(C₁₋₄alkyl), SH, S—C₁₋₄alkyl, NO₂, CF₃, OCF₃ and halo. 19.The compound according to claim 18, wherein Ar is an unsubstitutedphenyl group or phenyl group substituted with 1-2 substituentsoptionally selected from the group consisting of OH, OCH₃, NH₂, NHCH₃,N(CH₃)₂, SH, SCH₃, CF₃, OCF₃ and halo.
 20. The compound according toclaim 19, wherein Ar is selected from the group consisting of phenyl and3,4-dihydroxyphenyl.
 21. The compound according to claim 1, selectedfrom the group consisting of:(E,E)-2-(benzylamido)-3-styrylacrylonitrile (CR1);(E,E)-2-(benzylamido)-3-(3,4-dimethoxystyryl)acrylonitrile (CR2);(E,E)-2-(benzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR3); (E,E)-2-(benzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile (CR4);(E,E)-2-(phenylethylamido)-3-(3,4-dimethoxystyryl)acrylonitrile (CR5);(E,E)-2-(phenylethylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR8);(E,E)-2-(phenylpropylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR9);(E,E)-2-(3,4-dihydroxybenzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR11);(E,E)-2-thioacetamido-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR12); (E,E)-2-acetamido-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR13); (E,E)-2-carboxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR14); (E,E)-2-carbomethoxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile (CR15);(E,E)-2-acetamido-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile(CR16);(E,E)-2-acetamido-3-(3,4-dihydroxystyryl)acrylonitrile (CR17);(E,E)-2-(benzylamido)-3-(3,4-bis(t-butyldimethylsilyloxystyryl))acrylonitrile(CR18); (E,E)-2-(3,4 dihydroxybenzylamido)-3-styrylacrylonitrile (CR19);(E,E)-2-(3,4dihydroxybenzylamido)-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile(CR20); (E,E)-2-(3,4dihydroxybenzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile (CR21);(E,E)-2-(β-ethanolamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR24); (E,E)-2-(benzylamido)-3-(4-nitrostyryl)acrylonitrile (CR27);(E,E)-2-(3,4-dihydroxybenzylamido)-3-(4-nitrostyryl)acrylonitrile(CR28);and (E,E)-2-(1-amino-2,2-dicyanoethenyl)-3-(4-nitrostyryl)acrylonitrile(CR29).
 22. The compound according to claim 21, selected from the groupconsisting of: (E,E)-2-(benzylamido)-3-styrylarylonitrile (CR1);(E,E)-2-(benzylamido)-3-(3,4-dimethoxystyryl)acrylonitrile (CR2);(E,E)-2-(benzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR3); (E,E)-2-(benzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile (CR4);(E,E)-2-(phenylethylamido)-3-(3,4-dimethoxystyryl)acrylonitrile (CR5);(E,E)-2-(phenylpropylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR9);(E,E)-2-(3,4-dihydroxybenzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR11);(E,E)-2-thioacetamido-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR12); (E,E)-2-acetamido-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR13); (E,E)-2-carboxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR14);(E,E)-2-carbomethoxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR15); (E,E)-2-acetamido-3-(3,4-dihydroxystyryl)acrylonitrile (CR17);(E,E)-2-(3,4 dihydroxybenzylamido)-3-styrylacrylonitrile (CR19);(E,E)-2-(3,4 dihydroxybenzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile(CR21); and(E,E)-2-(β-ethanolamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR24).
 23. The compound according to claim 22, selected from the groupconsisting of:(E,E)-2-(benzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile (CR4);(E,E)-2-(3,4-dihydroxybenzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR11); (E,E)-2-acetamido-3-(3,4-dihydroxystyryl)acrylonitrile (CR17);(E,E)-2-(3,4 dihydroxybenzylamido)-3-styrylacrylonitrile (CR19);(E,E)-2-(3,4 dihydroxybenzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile(CR21); and(E,E)-2-(β-ethanolamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR24).
 24. The compound(E,E)-2-(benzylamido)-3-(3,4-dihydroxystyryl)acrylonitrile (CR4). 25.The compound(E,E)-2-(3,4-dihydroxybenzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR11).
 26. The compound(E,E)-2-(3,4-dihydroxybenzylamido)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR11).
 27. A composition comprising a compound according to claim 1 inadmixture with a pharmaceutically acceptable diluent or carrier.
 28. Amethod of modulating cell proliferation comprising administering aneffective amount of a compound of claim 23 to modulate cellproliferation to a cell or animal in need thereof.
 29. A method ofinhibiting cell proliferation comprising administering an effectiveamount of a compound of claim 23 to inhibit cell proliferation to a cellor animal in need thereof.
 30. The method of claim 29, wherein the cellproliferation that is inhibited is cancer cell proliferation.
 31. Amethod of treating cancer comprising administering to an animal in needthereof an effective amount of a compound of claim
 23. 32. The method ofclaim 30 or 31 wherein said cancer is a hematopoietic cell cancer. 33.The method of claim 30 or 31 wherein said cancer is a leukemia, alymphoma, a myeloma or a carcinoma.
 34. The method of claim 33 whereinsaid leukemia is acute lymphoblastic leukemia, aggressive Philadelphia+leukemia, acute myelocytic leukemia, chronic myeloid leukemia, chroniclymphocytic leukemia or juvenile myelomonocyte leukemia.
 35. The methodof claim 34 wherein said leukemia is acute lymphoblastic leukemia.
 36. Amethod of modulating cell proliferation comprising administering aneffective amount of a compound capable of modulating cell proliferationaccording to claim 1 or a composition of claim 27 to a cell or animal inneed thereof.
 37. A method of inhibiting cell proliferation comprisingadministering an effective amount of a compound capable of inhibitingcell proliferation according to claim 1 or a composition according toclaim 27 to a cell or animal in need thereof.
 38. A method of inhibitingcancer cell proliferation comprising administering an effective amountof a compound capable of inhibiting cancer cell proliferation accordingto any one of claim 1 or a composition according to claim 27 to a cellor animal in need thereof.
 39. A method of treating cancer comprisingadministering an effective amount of a compound capable of inhibitingcancer cell proliferation according to claim 1 or a compositionaccording to claim 27 to a cell or animal in need thereof.
 40. A methodaccording to claim 38 or 39 wherein said cancer is a hematopoietic cellcancer.
 41. A method according to claim 38 or 39 wherein said cancer isa leukemia, a lymphoma, a myeloma or a carcinoma.
 41. A method accordingto claim 41 wherein said leukemia is acute lymphoblastic leukemia,aggressive Philadelphia+ leukemia, acute myelocytic leukemia, chronicmyeloid leukemia, chronic lymphocytic leukemia or juvenile myelomonocyteleukemia,
 43. A method according to claim 42 wherein said leukemia isacute lymphoblastic leukemia.